PA1-023

antibody from Invitrogen Antibodies

Targeting: NFAT5 KIAA0827, NF-AT5, NFATL1, NFATZ, OREBP, TONEBP

Provider product page for PA1-023

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [34]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [4]
Immunohistochemistry [2]
Other assay [29]

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Product number: PA1-023 - Provider product page
Provider: Invitrogen Antibodies
Product name: NFAT5 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-023 detects nuclear factor of activated T-cells 5 (NFAT5) from human and mouse cells as well as recombinant human protein.
Concentration: 1 mg/mL

NFAT5 protein structure - PA1-023 shown in red.

Supportive validation

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Experimental details: Immunofluorescent analysis of NFAT5 using anti-NFAT5 polyclonal antibody (Product # PA1-023) (shown in green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were then blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a rabbit polyclonal antibody recognizing NFAT5 (Product # PA1-023), at a dilution of 1:100 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of NFAT5 in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a NFAT5 polyclonal antibody (Product # PA1-023) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552). NFAT5 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of NFAT5 in MCF-7 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a NFAT5 polyclonal antibody (Product # PA1-023) at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552). NFAT5 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of NFAT5 in NIH-3T3 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a NFAT5 polyclonal antibody (Product # PA1-023) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552). NFAT5 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human brain tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a Rabbit Polyclonal Antibody recognizing NFAT5 (Product # PA1-023) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human skeletal muscle tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a Rabbit Polyclonal Antibody recognizing NFAT5 (Product # PA1-023) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Experimental details: Immunoprecipitation of NFAT5 was performed on U2OS cells. The antigen:antibody complex was formed by incubating 500 µg whole cell lysate with 3 µg of rabbit polyclonal antibody recognizing NFAT5 (Product # PA1-023) overnight on a rocking platform at 4°C. The immune-complex was captured on 50 µL Protein A/G Plus Agarose (Product # 20423). Captured immune-complexes were washed and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1%Tween for at least 1 hour. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Pierce Super Signal West Dura (Product # 34075).

Supportive validation

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Supportive validation

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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of NFAT5 was performed on U2OS cells. The antigen:antibody complex was formed by incubating 500 µg whole cell lysate with 3 µg of rabbit polyclonal antibody recognizing NFAT5 (Product # PA1-023) overnight on a rocking platform at 4øC. The immune-complex was captured on 50 µL Protein A/G Plus Agarose (Product # 20423). Captured immune-complexes were washed and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1%Tween for at least 1 hour. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Pierce Super Signal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Activation of the transgenic 9xNFAT-Luc reporter in fresh and mitogen-activated lymphocytes . A) Proliferating transgenic 9xNFAT-Luc T cells were exposed to increasingly hypertonic media in the presence of FK506, or stimulated with PMA plus ionomycin during 24 hours. Left panel: luciferase activity (RLU) shown is the mean +- S.E.M of five independent experiments. Right panel: NFAT5 and pyruvate kinase (protein loading control) were detected by Western blotting. One representative experiment is shown out of three performed independently. B) Luciferase activity in 9xNFAT-Luc transgenic thymocytes exposed to increasingly hypertonic media in the presence of FK506, or stimulated with PMA plus ionomycin during 24 hours. Mean +- S.E.M of three independent experiments is shown. C) Luciferase activity in 9xNFAT-Luc transgenic splenocytes stimulated during 24 hours with hypertonicity or PMA plus ionomycin, either immediately after their purification, or after a 24-hour preactivation with concanavalin A plus IL2. One representative experiment is shown out of three performed independently.

Supportive validation

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Experimental details: Figure 5 Sensitivity of NFAT5 transcriptional activity and expression to pharmacological inhibitors . A) Transgenic 9xNFAT-Luc splenocytes preactivated during 24 hours with concanavalin plus IL2 were treated with hypertonicity (400 mOsm/kg, 24 hours) in the absence or presence of FK506 (100 nM), SB203580 or SB202190 (both at 10 muM), PD98059 (10 muM), H89 (2 muM), wortmannin (0.5 muM), or LY294002 (LY). Luciferase activity in the upper panel (mean +- S.D of three independent experiments) is shown as percentage of the activity in the absence of inhibitors (100%). Western blotting in the lower panelshows NFAT5 and pyruvate kinase expression. B) Transgenic MEF were stimulated for 8 or 24 hours with hypertonicity (460 mOsm/kg) in the absence or presence of SB203580 (10 muM) or LY294002 (LY). Luciferase activity is shown is the upper panel (mean +- S.E.M of three independent experiments). NFAT5 and pyruvate kinase expression are shown in the lower panel.

Supportive validation

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Experimental details: Figure 2 Induction of p53, p21 and GADD45 in proliferating NFAT5 -/- lymphocytes exposed to hypertonic stress. A) Western blot shows the time course of p53-Ser15 phosphorylation, accumulation of total p53 and induction of p21 in NFAT5 +/+ and NFAT5 -/- cells in response to hypertonicity. The experiment shown is representative of three independently performed (see Fig. S4 ). B) RNA was isolated from NFAT5 +/+ and NFAT5 -/- proliferating T cells that were either maintained in isotonic conditions (300 mOsm/kg) or switched to hypertonic medium (500 mOsm/kg) for 8 and 24 hours. Relative mRNA abundance for each GADD45 isoform was determined by RT-qPCR and values were normalized to their respective L32 mRNA levels (bars are mean+-SEM of four independent experiments).

Supportive validation

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Experimental details: Figure 4 Expression of cyclins in proliferating NFAT5 -/- T cells upon exposure to hypertonic conditions. A) Expression of cyclins D3, E1, A2 and B1 was analyzed by Western blot in lysates of proliferating NFAT5 +/+ and NFAT5 -/- T cells after 8 and 24 hours of hypertonicity treatment. Pyruvate kinase (PyrK) is shown as protein loading control. The result is representative of at least four independent experiments (see Fig. S5 ). B) mRNA abundance of cyclins was analyzed by RT-qPCR in proliferating NFAT5 +/+ and NFAT5 -/- T cells subjected to hypertonicity for 8 and 24 hours. Values were normalized to L32 mRNA levels in each respective sample (bars are the mean+-SEM of four independent experiments; * = p

Supportive validation

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Experimental details: Figure 5 Expression and activity of NFAT5 throughout the cell cycle. A) Proliferating NFAT5 +/+ T cells cultured during 24 hours in isotonic (300 mOsm/kg) or hypertonic (500 mOsm/kg) conditions were labeled with Hoechst 33342 and sorted according to DNA content as G0/G1, S or G2/M phase. Cell cycle histograms in the left panel show the efficiency of the sorting. Sorted cells were lysed and equal amounts of protein from each lysate were analyzed by Western blot with anti-NFAT5 antibody. NFAT1 and anti-pyruvate kinase (PyrK) are shown as protein loading controls. The result is representative of three independent experiments. B) Transgenic 9xNFAT-Luc T cells were labeled with Hoechst 33342 and sorted after 8 and 24 hours of exposure to hypertonicity. Sorted cells were split for Western blot and luciferase activity assays. A representative Western blot analysis (upper panel) is shown of three independently performed. Pyruvate kinase (PyrK) is shown as protein loading control. 9xNFAT-Luc reporter activity normalized to endogenous LDH is shown in the lower panel. Results are the mean+-SEM of three independent experiments.

Supportive validation

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Experimental details: Figure 6 Effect of pathologic hypertonicity on the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in NFAT5 -/- T cells. A) Splenocytes were induced to proliferate with anti-CD3/CD28 antibodies plus IL-2 in isotonic or moderately hypertonic medium. Cultures were harvested at the indicated time points and depleted of B cells. RNA was isolated and analyzed by RT-qPCR. All values were normalized to each respective L32 mRNA level. Graphs represent the mRNA abundance of the indicated gene products in hypertonic relative to isotonic conditions. Values correspond to the mean+-SEM of three independent experiments (* = p

Supportive validation

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Experimental details: Figure 1 NFAT5 regulates Wnt/ beta -catenin signaling. ( a ) Effects of NFAT5 on TCF/ beta -catenin reporter activity. HEK293 and Caco-2 cells were co-transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid together with either empty vector, Myc-NFAT5 or Flag- beta -catenin alone or Myc-NFAT5 plus Flag- beta -catenin, and the transfected cells were incubated for 48 h (upper panels). HEK293 and Caco-2 cells were transfected with control siRNA or siRNA targeting NFAT5. After a 48-h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after retransfection, cells were incubated with control-conditioned medium or Wnt3a-conditioned medium for additional 12 h (middle panels). Cells were harvested and luciferase activity was measured in the crude cell lysates, as described in Materials and Methods. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. (Data represent mean+-S.D.; * P

Supportive validation

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Experimental details: Figure 2 NFAT5 interacts with beta -catenin. ( a and b ) NFAT5 interacts with beta -catenin. HEK293 cells were co-transfected with Myc-tagged NFAT5 and empty vector or Flag-tagged beta -catenin, and incubated for 48 h. After anti-Flag ( a ) or anti-Myc ( b ) immunoprecipitation, the presence of NFAT5 and beta -catenin were analyzed using anti-Myc and anti-Flag antibodies, respectively. Western blot was performed on cell lysate as a control. ( c ) Interaction between endogenous NFAT5 and beta -catenin. Endogenous NFAT5 was immunoprecipitated from HEK293 or Caco-2 cells, and Western blot was performed on the eluate. IgG was used as a negative control. ( d ) Schematic diagram of beta -catenin deletion constructs. The yellow boxes are armadillo repeats. ( e ) C-terminal-transactivating domains of beta -catenin bind NFAT5. HEK293 cells were co-transfected with Myc-tagged beta -catenin deletion mutants. Myc-tagged beta -catenin mutants were immunoprecipitated using an anti-Myc antibody. Expression of NFAT5 was analyzed by western blot with an anti-NFAT5 antibody. The expression of these beta -catenin mutants was analyzed by western blot with an anti-Myc antibody. Western blot was performed on cell lysate as a control. ( f ) Schematic diagram of the wild-type and mutant derivatives of GST- tagged beta -catenin. ( g ) NFAT5 directly interacts with beta -catenin. HEK293 cells were transfected with Myc-NFAT5 and after 48 h incubation, lysate was collected and incubated with the GST pro

Supportive validation

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Experimental details: Figure 5 NFAT5 regulation of intestinal cell differentiation. ( a ) Knockdown of NFAT5 attenuated NaBT-induced IAP and sucrase activities in Caco-2 cells. Caco-2 cells were transfected with control siRNA or siRNA targeting NFAT5. After a 24-h incubation, transfected cells were treated with NaBT (5 mM) for additional 72 h. Cells were lysed and alkaline phosphatase and sucrase activities were determined. (Data represent mean+-S.D.; * P

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Experimental details: Figure 11. Transcriptional targets of NFAT5 upon biomechanical stretch. Volcano plot analysis of transcriptome changes (fold expression level vs calculated probability value) in HUASMCs transfected with control or NFAT5 siRNA shows nearly 2000 differently regulated genes for P

Supportive validation

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Experimental details: 3 Figure NFAT-5 gene and protein expression after (a) tensile strain or (b) compressive strain of RAW264.7 macrophages during normal or high salt (NaCl) conditions for 4 h. (c) NFAT-5 gene and protein expression in RAW264.7 macrophages [wild type (Wt)] or NFAT-5-overexpressing macrophages (NFAT-5 over ). One representative immunoblot each. Data were analyzed by ANOVA followed by Holm-Sidak or Games-Howell multiple comparison tests. AU, arbitrary units; mRNA, messenger RNA; NFAT-5, nuclear factor of activated T cells-5; * P < 0.05; ** P < 0.01; *** P < 0.001.

Supportive validation

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Experimental details: Fig 7 Pharmacological inhibition of NCX activity blocks HS-boosted MPhi activity. (A) Immunoblotting and densitometry of NFAT5 6 h after LPS +- HS in RAW264.7 MPhis +- KB-R pretreatment ( n = 3; paired t test; * p < 0.05). (B) Nos2 levels in RAW264.7 MPhis 4 h after LPS +- HS +- KB-R pretreatment (means +- SD; n = 10; Student t test; * p < 0.05). (C) Immunoblotting and densitometry of NFAT5 in RAW264.7 MPhis 4 h after LPS +- HS +- NiCl 2 pretreatment ( n = 4; paired t tests; * p < 0.05). (D) Nos2 levels in RAW264.7 MPhis 4 h after LPS +- HS +- NiCl 2 pretreatment (means +- SD; n = 6; Student t test + Welch correction; * p < 0.05). (E) Immunoblotting and densitometry of NFAT5 in RAW264.7 MPhis 4 h after LPS +- HS +- SEA pretreatment ( n = 5; paired t test; * p < 0.05). (F) Nos2 in RAW264.7 MPhis 4 h after LPS +- HS +- SEA pretreatment (means +- SD; n = 10-12; Mann-Whitney test; * p < 0.05). (G) RFP-GFP-mLC3 RAW264.7 MPhis were infected with Escherichia coli +- HS +- SEA pretreatment. Representative images 2 h after infection (RFP: red; GFP: green; scale bar: 10 mum). (H) Relative E . coli load at 2 h after infection of RAW264.7 MPhis +- HS +- SEA pretreatment (means +- SD; n = 12; Student t tests; * p < 0.05). For numerical raw data, please see S1 Data . For raw immunoblots, please see S1 Blots . CFU, colony forming unit; GFP, green fluorescent protein; HS, high salt; KB-R, KB-R7943 mesylate; LPS, lipopolysaccharide; mLC3, microtubule-associated protein 1 light chain 3; MPhi,

Supportive validation

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Experimental details: Fig 9 Slc8a1 silencing abrogates HS-augmented MPhi activity. (A) ns siRNA and Slc8a1 siRNA RAW264.7 were treated with LPS +- HS. Representative NFAT5 and ACTIN immunoblot 4 h after stimulation from two experiments. (B) As in (A), but Nos2 in BMDMs 4 h after LPS +- HS (means +- SD; n = 6; Mann-Whitney test; * p < 0.05). (C) As in (B), but Delta nitrite HS LPS-LPS after 24 h (means +- SD; n = 8; Student t test). (D) ns siRNA and Slc8a1 siRNA-treated RFP-GFP-mLC3 RAW264.7 MPhis were infected with E . coli +- HS. Representative images 2 h after infection from three experiments (RFP: red; GFP: green; scale bar: 10 mum). (E) ns siRNA and Slc8a1 siRNA-treated BMDMs were infected with E . coli +- HS. E . coli load at 2 h infection (means +- SD; n = 12; Student t tests; * p < 0.05). For numerical raw data, please see S1 Data . For raw immunoblots, please see S1 Blots . BMDM, bone marrow-derived MPhi; CFU, colony forming unit; GFP, green fluorescent protein; HS, high salt; LPS, lipopolysaccharide; mLC3, microtubule-associated protein 1 light chain 3; MPhi, monocyte/macrophage-like cell; NFAT5, nuclear factor of activated T cells 5; Nos2 , nitric oxide synthase 2; NS, normal salt; ns, nonsilencing; n.s., not significant; RFP, red fluorescent protein; siRNA, small interfering RNA; Slc8 , solute carrier family 8.

Supportive validation

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Experimental details: 3 FIGURE K14-IL-17A ind/+ mice utilize urea- and water transport mechanisms in kidney and skin to successfully prevent body dehydration. A, Urea transporter A1 (UT-A1) and tonicity enhancer binding protein (TonEBP) protein expression in the kidney of IL-17A ind/+ (n = 6) and K14-IL-17A ind/+ mice (n = 6). B, Urea content in the kidney of IL-17A ind/+ (n = 12) and K14-IL-17A ind/+ mice (n = 12). C, Expression of Aquaporin 2 mRNA in kidneys of IL-17A ind/+ (n = 5) and K14-IL-17A ind/+ mice (n = 5). D, UT-A1 and TonEBP protein expression in the skin of IL-17A ind/+ (n = 6) and K14-IL-17A ind/+ mice (n = 6). E, Urea content in skin of IL-17A ind/+ (n = 6) and K14-IL-17A ind/+ mice (n = 6). F, Expression of Aquaporin 3 mRNA in skin of IL-17A ind/+ (n = 5) and K14-IL-17A ind/+ mice (n = 5). G, Immunofluorescence stained skin sections from healthy IL-17A ind/+ control and psoriatic K14-IL-17A ind/+ mice (red: TonEBP; green: CD-45; blue: DAPI). Scale bars equal 50 um. (H) Tissue water content in skin, carcass and whole body of IL-17A ind/+ (n = 7) and K14-IL-17A ind/+ (n = 7) animals. For additional data about total body and tissue water see Online Table S2. Data analysis by Student's t test for independent samples. Data are shown as mean +- SEM. * P < .05; + P < .01; ++ P < .001

Supportive validation

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Experimental details: Figure 7 Representative Western blots and relative content of NFAT5 in HUVEC exposed to normal- and elevated-sodium medium for 3, 6 and 24 h. The content of proteins in control cells was taken as 1.0. Data obtained in three independent experiments are reported as means +- S.E. Figure 7

Supportive validation

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Experimental details: Figure 8 Representative Western blots and relative cytosol and nuclear content of NFAT5 and Lamin B1 in HUVEC exposed to normal- and elevated-sodium medium for 1, 3, 6 and 24 h. The content of proteins in control cells was taken as 1.0. Data obtained in three independent experiments are reported as means +- S.E. Figure 8

Supportive validation

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Experimental details: Figure 6 TNF production under hypertonic conditions involves p38 and NFAT5. ( a ) REH cells were cultured in the presence and absence of 50 mM NaCl for 16 h. Cells were washed, lysed and subsequently analyzed using western blotting with antibodies specific for the indicated proteins. ( b ) REH cells were challenged with 1.25 mu M birinapant in the presence and absence of 50 mM NaCl for the indicated periods of time. Subsequently, cells were lysed and analyzed by western blot for the indicated proteins. The asterisks (*) in the phospho-p38, ERK and phospho-ERK blots indicate a defect in the CCD sensor of the western Blot imaging system. All samples were run on the same gel, no gels were sliced. ( c - e ) REH cells were challenged with 2.5 mu M birinapant under isotonic (medium) or hypertonic (+50 mM NaCl) conditions in the presence and absence of the p38 inhibitors BIRB796, PH797804 and SB203580 (5 mu M). Shown are data points and mean+-S.E.M. from four independent experiments. ( f ) REH cells were challenged with 50 mM NaCl for 210 min in the presence and absence of the p38 inhibitors BIRB796, PH797804 and SB203580 (5 mu M). Tnf mRNA induction was analyzed by qPCR. Data points of three independent experiments are shown. Biri, birinapant

Supportive validation

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Experimental details: Figure 7. NFAT5 is required for HS-facilitated targeting of intracellular E. coli to autolysosomes. (a to d) As in Figure 1(f ), but non-silencing siRNA (ns siRNA) and Nfat5 -specific siRNA ( Nfat5 siRNA)-treated RAW264.7 MPhi were used. (a) Nfat5 mRNA levels (means +- s.e.m; n = 6; Student's t test or Mann-Whitney test, * p < 0.05). (b) Immunoblotting of NFAT5 and ACT. (c) As (a), but Atg7 mRNA levels (means +- s.e.m; n = 6; Student's t test +- Welch correction). (d) As (a), but Ulk1, Becn1, Atg4c and Atg2a mRNA levels (means +- s.e.m; n = 6; Student's t test +- Welch correction; * p < 0.05). (e) Immunoblotting and densitometry of MAP1LC3B/LC3 and ACT 1/2 h after infection (n = 6; paired Student's t test or Wilcoxon signed rank test; * p < 0.05). (f) As in Figure 1(f ), but ns siRNA- and Nfat5 siRNA-treated RFP-GFP-mLC3 RAW264.7 MPhi were used. RFP+ and RFP+GFP+-puncta were counted per cell. At least 290 cells from two independent experiments (bars: median scores; Kruskal Wallis test with Dunn's Multiple Comparison Test, * p < 0.05). (g) As Figure 1(f ), but RAW264.7 MPhi were used and stained with LysoTracker. Colocalization of sfGFP- E. coli and LysoTracker per cell. At least 101 cells from at least two independent experiments (bars: median scores; Kruskal Wallis test with Dunn's Multiple Comparison Test, * p < 0.05). (h) As in Figure 1(a ), but ns siRNA- and Nfat5 siRNA-treated RAW264.7 MPhi were used. Intracellular E. coli load in CFU relative to mean CFU under NS condit

Supportive validation

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Experimental details: Figure 8. Blunted AKT and MTOR activation does not account for HS-augmented antibacterial activity. (a) As in Figure 1(f ), RAW264.7 MPhi were pretreated +- 1 uM Torin1 and infected +- HS. Cells were harvested 1/2 h post infection. Immunoblots of p-AKT, AKT, MAP1LC3B and ACT levels are displayed. (b) As in (a), but 2 h post infection, NFAT5 and ACT levels were assessed. (c) As in (b), but RFP-GFP-mLC3 RAW264.7 MPhi were used. A representative confocal image out of two experiments is shown. Scale bar = 10 um. (d) As in (c), but sCFP3A- E. coli were used. Rate of E. coli- sCFP3A / RFP-colocalization per cell. At least 184 MPhi from two independent experiments (bars: median scores; Mann Whitney test; * p < 0.05). (e) As in (b), but intracellular E. coli load is displayed 2 h after infection in CFU relative to mean CFU under NS conditions (means +- s.e.m; n = 23-24; ANOVA with Bonferroni's test; * p < 0.05).