44-696G

antibody from Invitrogen Antibodies

Targeting: PLCG1 NCKAP3, PLC-II, PLC1, PLC148, PLCgamma1

Provider product page for 44-696G

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [18]
Comments [0]
Validations
Immunocytochemistry [1]
Other assay [10]

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Product number: 44-696G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-PLCG1 (Tyr783) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

PLCG1 protein structure - 44-696G shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Inhibition of Csk AS in primary murine T cells induces hyperactivation of SFKs and phosphorylation of TCR proximal signaling molecules. ( a ) Wild-type (WT) or Csk AS (AS) thymocytes or ( b ) peripheral CD4 + T cells were treated for the indicated times with 10 uM 3-IB-PP1 and analyzed by immunoblotting. ( c ) Wild-type (WT) or Csk AS (AS) thymocytes were treated for 3 min with vehicle (DMSO), 3-IB-PP1 or anti-CD3epsilon and analyzed by immunoblotting. ( d ) Wild-type (WT) or Csk AS (AS) thymocytes were treated for 3 min with vehicle (DMSO), 10uM 3-IB-PP1 or 20ug/mL anti-CD3epsilon, then lysed and immunoprecipitated with anti-CD3 zeta antibody. Immunoprecipitates were analyzed by immunoblotting. ( e ) Wild-type (WT) or Csk AS (AS) thymocytes were treated for 3 min with vehicle (DMSO), 10uM 3-IB-PP1 or 20ug/mL anti-CD3epsilon and analyzed by immunoblotting. All data are representative of 3 independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. Inhibiting Csk increases the magnitude of ligand-induced TCR signaling and reduces the threshold for TCR activation. ( A ) Purified Csk AS (AS) or wildtype (WT) CD4 + T cells stimulated for 2 min with 1 mug/ml, 5 mug/ml or 10 mug/ml anti-CD3epsilon antibody in the presence of DMSO or 10 muM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylation of the activation loop tyrosine of Lck and Fyn (Src pY416 antibody) and the inhibitory tyrosine of Lck (Lck pY505), phosphorylated ZAP-70 (ZAP70 pY319), LAT (LAT pY132) and PLC-gamma1 (PLCgamma1 pY783), as well as total actin (loading control). Data are representative of at least three independent experiments. ( B ) Purified total Csk AS (AS) or wildtype (WT) T cells stimulated with the indicated dose of anti-CD3epsilon antibody for 2 min in the presence of DMSO or 5 muM, 1 muM or 0.4 muM 3-IB-PP1 were analyzed for phosphorylated ERK (p-ERK) by phosphoflow. Histograms were gated on CD4 + cells. Data are representative of at least three independent experiments for AS cells and two independent experiments for WT cells. DOI: http://dx.doi.org/ Figure 1--figure supplement 1. Inhibiting Csk during TCR stimulation prolongs TCR signals. ( A ) Purified Csk AS CD4 + T cells stimulated for 2, 5 or 10 min with anti-CD3epsilon antibody in the presence of DMSO or 10 muM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylated ZAP-70, LAT and PLC-gamma1 as well as total GAPDH (loading control). Data are representative of a

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Calcium flux and Erk phosphorylation are restored by altering the actin cytoskeleton following Csk AS inhibition. ( a ) Csk AS (AS) thymocytes were stimulated with vehicle (DMSO), 10uM 3-IB-PP1, 20ug/mL anti-CD3epsilon, 10uM cytochalasin D (CytoD) alone or with 10uM 3-IB-PP1 for 5 min, and total inositol phosphate content was measured (mean, +- S.E.M., n = 3 (technical replicates), P -values from paired two-tailed student's t -test). ( b ) Wild-type (WT) or Csk AS (AS) thymocytes were stimulated with vehicle (DMSO: gray), 10uM 3-IB-PP1 (magenta), 20ug/mL anti-CD3epsilon (blue), 10uM cytochalasin D (CytoD: black) alone or with 10uM 3-IB-PP1(green). Ratiometric assessment of intracellular calcium of CD4 + CD8 + thymocytes over time is shown. ( c ) Wild-type (WT) or Csk AS (AS) thymocytes were stimulated for 2 min as in b , then analyzed for pErk content. Histograms are gated on CD4 + CD8 + thymocytes. ( d ) Wild-type (WT) or Csk AS (AS) thymocytes were treated for 3 min as in a and analyzed by immunoblotting. ( e ) Wild-type (WT) or Csk AS (AS) splenocytes were stimulated as in b . Ratiometric assessment of intracellular calcium of CD4+ splenocytes over time is shown. ( f ) Wild-type (WT) or Csk AS (AS) splenocytes were stimulated for 2 min as in b , then analyzed for pErk content. Histograms are gated on CD4 + splenocytes. Shaded histogram is DMSO control. ( g ) Wild-type (WT) or Csk AS (AS) thymocytes or ( h ) purified T cells were stimulated for 2 min as in b ,

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Pak2 plays a key role in functional maturation of CD4SP thymocytes. ( A ) Defects in mRNA expression of KLF2 in mature CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice. KLF2 mRNA levels in semi-mature and mature CD4SP thymocytes relative to Pak2 F/F DP thymocytes (left panel, mean +- SD of triplicates, results are representative of three independent experiment); KLF2 mRNA levels in mature Pak2 F/F ;Cd4 -Cre CD4SP thymocytes relative to mature Pak2 F/F CD4SP thymocytes (middle panel, mean +- SEM; each dot represents one mouse, n = 3); KLF2 mRNA levels in semi-mature Pak2 F/F ;Cd4 -Cre CD4SP thymocytes relative to semi-mature Pak2 F/F CD4SP thymocytes (right panel, mean +- SEM; each dot represents one mouse, n = 3). ***, p=0.0001; **, 0.001

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Inflammatory priming is required for SFK activation to produce downstream signaling in the absence of receptor clustering. ( A ) Csk AS BMDMs were incubated 12-16 hr in non-priming medium or in medium containing 25 U/ml IFN-gamma. Signal transduction after 3-IB-PP1 or zymosan dep treatment was assessed by immunoblotting with antibodies specific to activating phosphorylation sites of DAP12, Syk, LAT, PLCgamma2, PLCgamma1, and Erk (arrow). Total Erk1 is shown as a loading control. ( B ) Signal transduction in the PI3K/Akt and JNK pathways was assessed by immunoblotting with antibodies specific for phosphorylated c-Cbl, PI3K, Akt (arrow), and JNK (arrow). Total JNK is shown as a loading control. NFAT1 activation was assessed by the faster migration of cellular NFAT1 upon dephosphorylation. See Figure 5--figure supplement 1 for further characterization of signaling in primed and unprimed cells. DOI: http://dx.doi.org/ Figure 5--figure supplement 1. Further characterization of signaling in primed and unprimed BMDMs. ( A ) Treatment of IFN-gamma-primed and unprimed Csk AS BMDMs with 3-IB-PP1 led to activating phosphorylation of the cytoskeleton-associated SFK substrates HS1, Vav, and FAK. ( B ) Comparison of background Erk phosphorylation in unprimed and IFN-gamma-primed Csk AS BMDMs with pulse-spin only. DOI: http://dx.doi.org/

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Imparied Puma activation contributes to TKI resitance SCLL. Western blot analysis of PUMA protein levels in the murine SCLL BBC1 cell line ( A , above) or human KG1 cell line, after treatment at the indicated concentrations of either the ponatinib or BGJ398 FGFR1 inhibitors, show a dose dependent increase in the parental cells, while no changes are seen in the resistant cells that carry FGFR1 mutations in the inhibitor binding site. The same effect is seen in in cell lines CEP2A and ZNF112, which are resistant due to Pten deletions ( B , above). RT-PCR analysis in all four cell lines shows that transcription levels of PUMA increases significantly in the cells treated ( A , B , below) with the FGFR1 inhibitors at the maximum concentrations shown in A and B (above). Flow cytometric analysis of BBC1 and CEP2A cells shows an increase in 7AAD/Annexin V+ cells in the parental cells treated with BGJ398 at the IC50, but no changes are observed in the resistant cells ( C ). Western blot analysis ( D ) of signaling intermediates demonstrates consistent Akt activation and Puma suppression in the resistant cells (see text) and phosphorylation of Foxo3a is suppressed in parental, but not in the resistant cells treated with BGJ398. Student's t -test was used for statistical comparison between groups. ** p < 0.01; *** p < 0.001; ns not significant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure EV3 VEGF-A/VEGFR2 downstream signalling in vitro and in vivo A, B HDMEC, non-transfected (NT) or transfected with non-targeting (c/cntrl) or PALD1 -targeting siRNA (KD #1, KD #2), were treated with VEGF-A for the indicated time periods. Immunoblots of cell lysates to determine protein levels of ACTIN, Paladin, pTyr416, and pTyr527 of Src family kinases (SFK) and total Src (A), pSer473 AKT and total AKT (B), are shown (left) and their quantification (right). Mean +- SEM, two-way ANOVA, n = 3 biological replicates. C Immunoblotting of total heart lysates from Pald1 +/+ and Pald1 -/- mice, tail vein injected with VEGF-A or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, phospholipase Cgamma (PLCgamma), Erk1/2, and Akt, and beta2-microglobulin (beta2-MG, loading control). Part of the same blot is also shown in Fig 3 . Quantification of pY783 PLCgamma normalized to PLCgamma ( n = 4 biological replicates) and pS473 Akt normalized to Akt ( n = 5 biological replicates), Mean +- SEM, two-way ANOVA. SSSS D Pald1 +/+ and Pald1 -/- mice were tail vein injected with VEGF-A or PBS for the indicated time periods. Heart lysates were blotted to determine the phosphorylated and total levels of Src family kinases (SFK). pY416 SFK normalized to pY527 SFK. Mean +- SEM, two-way ANOVA, n = 5 biological replicates. Data information: * P < 0.05, ** P < 0.01. Source data are available online for this figure.