51-1300

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Expression of SMAD2 was detected after transfection of HeLa cells with 50nM Silencer® SMAD2 siRNA (Product # 115714) (lane 3) or 50nM Silencer® Negative Control siRNA (lane 2) and untransfected (lane 1) using SMAD2 rabbit polyclonal antibody (Product # 51-1300, 2.5 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Relative density of the bands normalized to actin confirms silencing of SMAD2 expression and the specificity of SMAD2 rabbit polyclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Rat Testes (Lane 1), Mouse Testes (Lane 2), C2C12 (lane 3), MDA-MB-231 (lane 4) and MCF7 (lane 5). The blots were probed with Anti-SMAD2 Rabbit Polyclonal Antibody (Product # 51-1300, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 52 kDa band corresponding to SMAD2 was observed across cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of SMAD2 showing staining in the nucleus of paraffin-embedded mouse breast tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a SMAD2 polyclonal antibody (Product # 51-1300) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of SMAD2 showing staining in the nucleus of paraffin-embedded human breast tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a SMAD2 polyclonal antibody (Product # 51-1300) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of SMAD2 showing staining in the nucleus of paraffin-embedded mouse breast tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a SMAD2 polyclonal antibody (Product # 51-1300) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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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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Experimental details: Figure 4. Phospho-Smad2 and Smad4 regulate LM alpha3 subunit gene transcription. (A) Differential phosphorylation of Smads. Subconfluent (Subcfl.) or confluent cultures either untreated (control) or treated with 5 ng/ml TGF-beta1 for 6 h in the absence or presence of the TbetaR-I inhibitor SB431542 (+TGF-beta1 or + TGF-beta1+SB43, respectively) were analyzed by Western blotting for phospho-Smad2 (P-Smad2), total Smad2, P-Smad3, and total Smad3. (B) Phospho-Smad2 is localized to the nuclei after TGF-beta1 treatment in confluent cells. Untreated (control) or TGF-beta1-treated confluent cells (+TGF-beta1) were stained with antibodies against P-Smad2 (red) and LM-332 (beta3 subunit; green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 mum. (C) Smad 4 is also localized to the nuclei in subconfluent and TGF-beta1-treated confluent cells. Subconfluent MDCK cell cultures without added exogenous TGF-beta1 and confluent cultures either untreated (control) or treated with of TGF-beta1 in the presence or absence of SB431542 (SB) were stained with antibodies against Smad4 (green) and analyzed by confocal fluorescence microscopy. Nuclear staining with DAPI, blue. Bar, 10 mum. (D) Phosho-Smad2 and Smad4 form a complex dependent on TbetaR-I signaling. Confluent cultures treated with TGF-beta1 to induce LM-332 expression in the absence or presence of the TbetaR-I inhibitor SB431542 (+TGF or +TGF+SB43, respectively) were extracted in RIPA buffer. E

Supportive validation

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Experimental details: Figure 6. Mixed Smad complexes are expressed in mammary tumors but not normal mammary epithelium. (A-F) MDA-MB-231 xenograft tumors grown in the mammary fat pads of mice show expression for all three Smad complexes: (D) canonical BMP Smad, (E) canonical TGF-beta Smad and (F) mixed Smad complexes, whereas mammary epithelium, with a more normal histological appearance, shows expression of canonical Smad complexes (A, B), but not mixed Smad complexes (C). (G-L) A human breast tumor shows the expression of canonical and mixed Smads (J-L) whereas normal breast tissue from the same patient expresses the canonical TGF-beta Smad complexes (H) but no mixed Smad complexes (I). Arrowheads mark examples of positive rolling circle amplification (RCA) products in panels with detectable signals. Scale, 10 um.

Supportive validation

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Experimental details: Figure 3. Proximity ligation assay (PLA) detection of Smad complexes. (A) MDA-MB-231 cells were treated with TGF-beta or BMP2 for 1 hr and pSmad2, pSmad3, or pSmad1/5/9 were detected by western blotting using commercial antibodies. Loading control, beta-actin. (B) Xenograft tumors generated by injection of MDA-MB-231 cells into nude mice were stained with the antibodies used in (A) using peroxidase immunohistochemistry. Scale, 25 um. (C) Canonical Smad complexes were detected by pairing either rabbit (Rb) anti-Smad1/5/9 (left) or anti-Smad2/3 (right) with mouse (Ms) anti-Smad4. The primary (1deg) antibodies are detected by species-specific secondary (2deg) antibodies coupled to either a (+) or (-) oligonucleotide that will hybridize if in close proximity. Following ligation and amplification, the rolling circle amplification (RCA) products are detected by peroxidase-conjugated oligonucleotides and detected with Nova Red. (D) Mixed Smad complexes were detected by conjugating either a (+) or (-) oligonucleotide directly onto the Rb 1deg antibodies and detected as described in (C). (E-H) PLA for mixed Smad complexes in formalin-fixed, paraffin-embedded (FFPE) sections of MDA-MB-231 cells transfected with siRNAs to a non-targeting sequence (E, G) or Smad2/3 (F, H). Cells were treated with TGF-beta (5 ng/ml) for 45 min (G, H) or left untreated (E, F). TGF-beta induced the expression of mixed Smad complexes in cells with non-targeting siRNA (G vs E), but not in cells with Smad2/3 k

Supportive validation

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Experimental details: Figure 7. Human breast cancer tissues from a test tissue microarray (TMA) show different patterns of expression of Smad complexes. The proximity ligation assay was used to detect canonical BMP Smad (A, D, G), canonical TGF-beta Smad (B, E, H) and mixed Smad (C, F, I) complexes in a test TMA of human breast cancer tissues. Images in each row were acquired from similar regions of the tissue cores. A-C and D-F represent two different areas of the same cores, whereas G-I are from an independent core. Arrowheads mark examples of positive rolling circle amplification (RCA) products in panels with detectable signals. Scale, 10 um.

Supportive validation

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Experimental details: Figure 1 KRAS G12D potentiates TGFbeta-mediated cell growth inhibition of AR42J rat pancreatic acinar cells. ( A ) Immunoblot of RAS-GTP and total RAS on AR42J-WT and AR42J-KRAS G12D protein extracts. ( B ) Phase microscopy of AR42J-WT and AR42J-KRAS G12D cells 48 hours after TGFbeta treatment. Insets represent higher magnifications fields. Scale bar, 50 mum/L. NT, untreated. *Apoptotic cells. ( C ) Cell count of AR42J-WT and AR42J-KRAS G12D cells at indicated times after TGFbeta treatment. Representative experiment performed in duplicate (means +- standard deviation) is shown. ( D ) Immunoblot of SMAD proteins after 2-hour TGFbeta treatment.

Supportive validation

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Experimental details: Figure 6 Inducible TbetaRI CA expression in pancreatic acinar cells after birth results in SMAD pathway activation in vivo . ( A ) Breeding strategy to express TbetaRI CA after birth in pancreatic acinar cells by using tamoxifen-inducible Pft1a-Cre ERT2 allele. Black arrows represent genotyping primers. Sizes of expected fragments are shown. ( B ) PCR on genomic DNA to assess excision of STOP signal in C ER R pancreas after tamoxifen injection. ( C ) RT-qPCR of TbetaRI C A and Serpine-1 on total RNA from pancreata prepared from mice treated with tamoxifen. Expression level in R mice was arbitrarily set at 1 (mean +- standard deviation; 2 independent experiments). ( D ) Immunoprecipitation of SMAD2/3 and Western blot analysis of P-SMAD2 (phospho-SMAD2) and total SMAD2. Total lysate was assessed for beta-tubulin and SMAD2. ( E ) RNAscope detection of TbetaRI CA and Smad7 mRNA on pancreatic sections from WT and C ER R mice. Scale bars, 100 mum. ( A-E ): WT, wild-type; R, [ LSL-TbetaRI CA ]; C ER R, [ Pft1a-Cre ERT2 ; LSL-TbetaRI CA ]. mag, magnification; TAM, tamoxifen.

Supportive validation

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Experimental details: Fig. 4 TGF- beta triggers Smad2 phosphorylation and transactivation. (A) 3TP luciferase reporter activity in SM10s cells treated with vehicle control or TGF- beta 2. Cells were transiently transfected with 3TP-lux and pRLSV40 using Metafectene. Twenty-four hours post-transfection, cells were treated with vehicle or TGF- beta 2 (5 ng/ml) for 72 hours. Luciferase activity was analyzed using the dual luciferase assay system. The 3TP-lux transactivation values were normalized to the constitutively active reporter (pRLSV40). (B) Western blotting analysis of Smad2, Smad3, and Smad4 in TGF- beta 2 or vehicle-treated SM10 cells. COS-7 cells were used as a positive control. (C) Western blotting analysis of Phospho-Smad3 in TGF- beta 2 or vehicle-treated SM10 cells. COS-7 cells transiently transfected with the plasmid, pXIX-Smad3 and treated with TGF- beta were used as a positive control. (D) Western blotting analysis of Phospho-Smad2 in the TGF- beta 2, Activin A, or vehicle-treated SM10 cells. (E) ARE luciferase reporter activity in SM10 cells transiently transfected with pCDNA3 control or pRK5-Smad2 and treated with vehicle control or TGF- beta 2. Cells were transiently transfected with ARE-lux, pLv-CMV-hFAST-1, and pRLSV40 using Metafectene. Twenty-four hours post-transfection, cells were treated with vehicle or TGF- beta 2 (5 ng/ml) for 72 hours. Luciferase activity was analyzed using the dual luciferase assay system. The ARE-lux transactivation values were normalized to the const

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Smad7 leads to higher Rac1 expression by repressing individual Smad proteins and CtBP1 binding to the SBE of the Rac1 promoter (a ) Rac1 mRNA levels (mean +- s.d.) in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. ( b ) Western blot analysis of GTP-bound Rac1 (GTP-Rac1) and total Rac1 in WT and Smad7 transgenic keratinocytes. Smad7 protein levels were determined by re-probing the tubulin western blot with an antibody to Smad7 (see an additional western blot and quantification in Supplementary Fig. 4a, b ). (c) The amount of Rac1 protein after knocking down Smad2, Smad3 or Smad4 individually in human keratinocytes (See Supplementary Fig. 4c-e for Smad knockdown efficiencies). siSamd2-4, siRNAs specific for Smad2-4. ( d ) ChIP assay for Smad2, Smad3, Smad4, and Smad7 binding to the SBE -1.5 kb site of the Rac1 promoter in keratinocytes from WT and Smad7 transgenic mice. ( e ) Rac1 luciferase reporter assay in mouse keratinocytes. n = 6 per group. siSmad7, siRNA specific for Smad7; Rac1-SBE, the SBE-1.5 kb site of the Rac1 promoter. Data are the mean +- s.d. ( f ) Activities (mean +- s.d.) of Rac1- luc reporters containing SBE (Rac1-SBE) or mutant SBE (Mut Rac1-SBE) in keratinocytes from WT and Smad7 transgenic mice. n = 6 per group. ( g ) Images of ChIP assays of CtBP1 binding to the SBE-1.5 kb site of the Rac1 promoter in keratinocytes from WT or K5.Smad7 mice. ( h ) ChIP-quantitative PCR (mean +- s.d.) of CtBP1 binding to the SBE in g in keratinocy

Supportive validation

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Experimental details: Figure 6 Compound 145 limits TGF-beta 1 -induced Smad-2 phosphorylation and nuclear translocation in MRC-5 cells. Lung fibroblasts were pre-incubated with the studied compounds (10 uM) for 1 h, and then TGF-beta 1 (5 ng/mL) was added for 1 h. ( A ) Representative immunoblot and densitometry analysis of p-Smad-2 in MRC-5 ( n = 4). The relative optical density (ROD) signal was normalized to the GAPDH control level. Each bar represents the mean value (+- S.E.M.). ( B ) Immunofluorescence staining for p-Smad-2 protein: MRC-5 were fixed, permeabilized, blocked, and incubated with anti-p-Smad-2 antibody followed by Alexa Fluor 546 conjugated antibody, nuclei were stained with the Hoechst 33342 dye. Microphotographs were taken using Leica DMi8 microscope with Thunder Imaging System, 40x objective, bar = 50 um. ( C ) Cells exhibiting strong nuclear p-Smad-2 signals were counted in 20 randomly selected fields of view and expressed as a percentage fraction in the entire MRC-5 population. The results were considered statistically significant at the p level of 0.05, against the control (#) and TGF-beta 1 (*) conditions.

Supportive validation

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Experimental details: Pan-PDE inhibitors attenuate TGF-beta-induced Smad-2 signaling in alveolar epithelial type II cells. A549 cells were cultured overnight and then serum-deprived for 24 h. 1 , 2 , 3 , or TEO (10 muM) was administrated 1 h before TGF-beta (5 ng/mL) treatment for an additional 1 h incubation. Representative immunoblots and densitometry analysis of p-CREB ( A ) and p-Smad-2 ( B ) in A549 ( n = 5). The relative optical density was normalized to the GAPDH control level. Horizontal lines represent the median with interquartile range (Kruskal-Wallis test with Dunn's post-hoc test). ( D ) Representative images (400x magnification) of A549 immunostained for p-Smad-2. Cells were fixed, permeabilized, blocked, and incubated with anti-p-Smad-2 antibody followed by an Alexa Fluor 546 conjugated antibody, and nuclei were co-stained with Hoechst 33342 dye. Scale bar = 50 um. ( C ) Cells exhibiting colocalization of p-Samd-2 and nuclear signal were counted in randomly selected fields of view and expressed as a percentage fraction in the entire A549 population. Horizontal lines represent the mean value (+-SD), n = 10 (ANOVA with Dunnett's post-hoc test). The results were considered statistically significant at the p -level of 0.05, against the control (#) and TGF-beta (*) conditions.

Supportive validation

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Experimental details: Fig. 6 TGF-ss2/TGFBR2 signaling regulates microglial homeostasis via CX3CR1. a Immunofluorescent staining of representative sections showing TGF-beta2 and NG2 immunoreactivities in the corpus callosum of wild-type mice. Arrows indicate double-labeled cells. Scale bar, 50 mum. b Measurement of TGF-beta2 protein levels was performed in the concentrated conditioned medium (CM) from primary NG2 glial cells using TGF-beta2 ELISA ( n = 6 for control medium, n = 7 for NG2 glia CM). Unpaired t test. ** P < 0.01. c Pre-incubation of NG2 CM with antibody against TGF-ss2 markedly reduces the mRNA levels of CX3CR1 and increases IL-1beta expression in primary cultured microglia. Unpaired two-tailed t test, ( n = 8-10). *** P < 0.001 for CX3CR1. Nonparametric test with Mann-Whitney test, ** P < 0.01 for IL-1beta. All data are expressed as mean +- SEM. d qPCR analysis shows that the increases in the expression of Tmem119 and CX3CR1 in microglial cell line BV2 cells were abolished upon addition of LY2109761, a TGFBR1/2 blocker. One-way ANOVA with Newman-Keul's post test (6-8 independent experiments). * P < 0.05, ** P < 0.01, **** P < 0.001. e Immunoblotting analysis reveals that the elevations in the levels of phopho-Smad2 evoked by TGF-beta2 are blocked by LY2109761 in BV2 cells. One-way ANOVA with Newman-Keul's post test ( n = 3 independent experiments). **** P < 0.001. f qPCR analysis shows that the decreases in the expression of CX3CR1 and Tmem119 in BV2 cells were induced upon transfect

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 Activation of TGF-beta2/TGFBR2 signaling attenuates LPS-induced neuroinflammation. a qPCR analysis showing the reduction of IL-1beta mRNA levels in primary cultured microglia treated with TGF-beta2 (5, 10 ng ml -1 , 4 h) in the presence or absence of LPS (50 ng ml -1 ). Unpaired t test ( n = 3 independent experiments). b , c Representative immunoblots showing the increases in the levels of IL-1beta and TNF-a protein induced by LPS were attenuated by TGF-beta2 treatment in BV2 cells ( n = 8 independent experiments). This process is accompanied by alterations in the levels of phospho-SMAD2 ( c ). n = 9 independent experiments. d qPCR analysis shows that the disruption of TGFBR2 signaling by LY2109761 exacerbates inflammatory response to LPS stimulation. BV2 cells were treated with LY2109761 (10 ng/ml) or vehicle DMSO for 2 h, followed by exposure to LPS (100 ng/ml) or saline for 6 h. Two-way ANOVA with Turkey's test ( n = 6 independent experiments). e , f Immunoblot analysis showing the increases in the levels of active-IL-1beta in BV2 cells with disruption of TGFBR2 signaling by LY2109761. f Quantification of data shown in e . Two-way ANOVA with Turkey's test. Data are expressed as mean +- SEM ( n = 3-6). * P < 0.05, ** P < 0.01, *** P < 0.001. g qPCR analysis of the striatum of wild-type mice that received stereotactic injection of lentivirus-TGFBR2 showed that the production of inflammatory mediators was attenuated by TGFbetaR2 overexpression

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5--figure supplement 1. Characterization of direct Nodal target genes. Left panel: binding peaks of Smad2 and its associated transcription factor FoxH1 at dome stage after injection of zebrafish Nodal Squint mRNA (red) or after treatment with the Nodal signaling inhibitor SB505124 (blue). Peaks called by the MACS algorithm are indicated (gray blocks). Middle panel: NanoString count levels after injection of recombinant mouse Nodal protein (red) or after SB505124 treatment (blue) in the absence (-cycloH) or in the presence (+cycloH) of the translation inhibitor cycloheximide. Right panel: time course induction of target genes after Nodal injection at 3.5 hpf (green) or 4.5 hpf (orange). In each case, (1) a specific Smad2 peak associated with a FoxH1 peak appears in the vicinity of the TSS upon Squint injection, (2) the Nodal-induced expression is not abolished by cycloheximide treatment, and (3) induction kinetics have similar trajectories independently of the stage of Nodal exposure, suggesting that these genes are direct targets of Nodal signaling. DOI: http://dx.doi.org/

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8--figure supplement 1. Characterization of co-regulated Nodal target genes. Left panel: binding peaks of Smad2 and the associated transcription factor FoxH1 at dome stage after injection of Squint mRNA (red) or after treatment with the Nodal signaling inhibitor SB505124 (blue). Peaks called by the MACS algorithm are indicated (gray blocks). Middle panel: NanoString count levels after mNodal injection (red) or after SB treatment (blue) in the absence (-cycloH) or the presence (+cycloH) of the translation inhibitor cycloheximide. Right panel: time course induction of target genes after Nodal injection at 3.5 hpf (green) or 4.5 hpf (orange). Although these genes have Smad2/FoxH1 binding sites, their induction is abolished in the presence of cycloheximide, suggesting that an additional transcriptional co-regulator controls their transcription. Moreover, these genes are delayed in their onset of expression. The length of the delay depends on the stage at which Nodal is applied. DOI: http://dx.doi.org/