Journal: Clinical Cancer Research

Article Title: Combined Src and Aromatase Inhibition Impairs Human Breast Cancer Growth In vivo and Bypass Pathways Are Activated in AZD0530-Resistant Tumors

doi: 10.1158/1078-0432.ccr-08-3127

Figure Lengend Snippet: Fig. 2. Effects of anastrozole and Src inhibitor AZD0530 on signaling and cell cycle regulators. Cells were grown without estrogen in 5% charcoal-stripped fetal bovine serum supplemented with androstendione (no drug) and treated with no additional drug, AZD0530 (SI), anastrozole (Ana), or both (Ana + SI) for 48 h and then assayed for (A) pSrc, Src, pAkt, Akt, pMAPK, and MAPK levels and (B) p27, cyclin E, cyclin D1, Cdk2, Cdk4, and β-actin levels. C, cyclin E was immunoprecipitated and complexes were resolved and immunoblotted for associated proteins. D, cyclin E immunoprecipitates were assayed for associated kinase activity as per Materials and Methods. Radioactivity in histone H1 substrate was quantitated. Mean ± SE % maximum kinase activity from four repeat assays.

Article Snippet: Western analysis of cyclin E, p27, Cdk2, Cdk4, cyclin D1, MAPK, pMAPK (MAPKpT202/Y204), Akt, pAkt (AktpT473), Src, and pSrc (Src-pY416) used the following: antibodies to MAPK, pMAPK, Akt, pAkt, Src, and pSrc were obtained from Cell Signaling; antibodies to p27 were from Transduction Laboratories; antibodies to cyclin E1 (monoclonal antibodies E172 and E12) were from E. Harlow (MGH); and antibodies to β-actin were from Sigma.

Techniques: Immunoprecipitation, Activity Assay, Radioactivity

Journal: Journal of Clinical Immunology

Article Title: A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature

doi: 10.1007/s10875-023-01602-8

Figure Lengend Snippet: Reduced protein expression and TCR signaling due to LCK C465R. A LCK and GAPDH immunoblots of whole cell lysates of patient or healthy control (HC) T-cell blasts unstimulated or stimulated with anti-CD3 (2 min), anti-CD3/CD28 (2 min), PMA/ionomycin (P/I, 5 min) or treated with the selective LCK-inhibitor A770041 (A77, 10 min). Orange arrowheads indicate LCK bands. B pZAP70, pSFK (pY416), pERK1/2 or GAPDH immunoblots, conditions as in A . Red arrowheads indicate pLCK bands, black arrowheads pFYN-T. Numbers below pERK1/2 blot indicate fold change pERK1/2 band intensity (HC unstimulated =1), intensity normalized to GAPDH loading (numbers below GAPDH blot; GAPDH signal intensity normalized to HC unstimulated) C LCK, HA-tag and GAPDH immunoblots of whole cell lysates of Jurkat E6.1, J.CaM1.6, and stable transduced cells line expressing either LCK WT or LCK C465R with or without on C-terminal HA-tag or transduced with pCDH containing only the expression cassette for the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR) (J.CaM EV). D Overlay of Ca 2+ -flux measurement in the indicated cell lines. Experiment representative of 3 independent experiments. E pZAP70, pSFK (pY416), pERK1/2 immunoblots in J.CaM1.6 cells transduced with pLJM1 containing LCK WT or LCK C465R and stimulated with either anti-CD3 (2, 5 or 15 min), anti-CD3/CD28 (2 or 5 min), PMA/ionomycin (5 min) or left untreated

Article Snippet: After a blocking step with 3% BSA TBS-T, immunoblotting was performed with the following antibodies: mouse anti-human FYN (clone E-3), mouse anti-human GAPDH (clone 6C5), mouse anti-human LCK (clone 3A5), mouse-IgGκ BP-HRP, rabbit-IgGκ BP-HRP, anti-mouse-IgG-HRP (all Santa Cruz Biotechnology) or rabbit anti-human pZAP70 pY319 (clone 65E4), rabbit anti-human polyclonal pSRC-family kinase (pSFK) pY416, rabbit anti-human pERK1/2 pT202/pY204 (clone 197G2), and rabbit anti-HA-tag (clone C29F4) (all Cell Signaling Technologies).

Techniques: Expressing, Western Blot, Control, Transduction

Journal: Nature Communications

Article Title: Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling

doi: 10.1038/s41467-018-05288-0

Figure Lengend Snippet: Analog amplifier model for the mechanism of coagonism. Nonstimulatory pMHC molecules recruit CD8-Lck into the immunological synapse. Preconcentrated Lck further phosphorylate the ITAMs of TCR-CD3ζ which are engaged with specific antigenic pMHC. Enhanced pCD3ζ provide more docking sites for Zap70 to be phosphorylated. Enhanced pZap70 signal amplifies the downstream TCR signaling pathway including the phosphorylation of Erk. pErk translocates into the nucleus and enhances the effector functional readouts of T-cell activation

Article Snippet: Anti-pLAT (Tyr191) (Cat# 3584S), P-p44/42 MAPK (T202/Y204) (Cat# 4370L), pSrc family (pY416) (Cat# 6943T), non-pSrc family (pY416) (Cat# 2102S), pSHP-1 (Cat# 8849S), pZap70 (pY319) (Cat# 2717S), Zap70 (Cat# 2709), pPLC-γ1 (pY783) (Cat# 2821S), α-tubulin (Cat# 3873S), GAPDH (Cat# 2118S) antibodies were from Cell Signaling Technologies.

Techniques: Phospho-proteomics, Functional Assay, Activation Assay

Journal: Nature Communications

Article Title: Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling

doi: 10.1038/s41467-018-05288-0

Figure Lengend Snippet: Coagonist pMHC enhance human T-cell response to antigens by increasing phosphorylation of molecules involved in proximal TCR signaling pathway. Human E183 CTL were stimulated by T-REx CHO cells panel for 5 or 30 min, lysed and the lysate was separated on NuPAGE Bis-Tris Gel. The filter was blotted with antibodies against pCD3ζ ( a ), pZap70 ( b ), pLAT ( c ), pPLC-γ1 ( d ), p-Erk1/2 ( e ), and pSHP-1 ( f ). The data are representative of three independent experiments. Uncropped western blotting images are presented in Supplementary Fig.

Article Snippet: Anti-pLAT (Tyr191) (Cat# 3584S), P-p44/42 MAPK (T202/Y204) (Cat# 4370L), pSrc family (pY416) (Cat# 6943T), non-pSrc family (pY416) (Cat# 2102S), pSHP-1 (Cat# 8849S), pZap70 (pY319) (Cat# 2717S), Zap70 (Cat# 2709), pPLC-γ1 (pY783) (Cat# 2821S), α-tubulin (Cat# 3873S), GAPDH (Cat# 2118S) antibodies were from Cell Signaling Technologies.

Techniques: Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling

doi: 10.1038/s41467-018-05288-0

Figure Lengend Snippet: Coagonist pMHC enhance the recruitment of pCD3ζ, pLck, and pZap70 into the immunological synapse. Different concentrations of E183-A2 monomer or GAG-A2 monomer were added to the lipid bilayers in a glass chamber slide. Totally, 10 5 E183 CTL were added into each well of the chamber and stimulated for 5 min. The CTL were fixed, permeabilized and stained with different antibodies to check the recruitment of pCD3ζ ( a , b ), pLck ( c , d ), and pZap70 ( e , f ) into the immunological synapse by TIRF microscopy. Anti-pSrc (Y416) antibody was used to detect active Lck (p-Y394). Statistical significance was determined by using unpaired two-sided Student’s t test and shown as mean ± s.d. (ns nonsignificant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data are representative of three independent experiments

Article Snippet: Anti-pLAT (Tyr191) (Cat# 3584S), P-p44/42 MAPK (T202/Y204) (Cat# 4370L), pSrc family (pY416) (Cat# 6943T), non-pSrc family (pY416) (Cat# 2102S), pSHP-1 (Cat# 8849S), pZap70 (pY319) (Cat# 2717S), Zap70 (Cat# 2709), pPLC-γ1 (pY783) (Cat# 2821S), α-tubulin (Cat# 3873S), GAPDH (Cat# 2118S) antibodies were from Cell Signaling Technologies.

Techniques: Staining, Microscopy

Journal: Nature Communications

Article Title: Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling

doi: 10.1038/s41467-018-05288-0

Figure Lengend Snippet: Analog amplifier model for the mechanism of coagonism. Nonstimulatory pMHC molecules recruit CD8-Lck into the immunological synapse. Preconcentrated Lck further phosphorylate the ITAMs of TCR-CD3ζ which are engaged with specific antigenic pMHC. Enhanced pCD3ζ provide more docking sites for Zap70 to be phosphorylated. Enhanced pZap70 signal amplifies the downstream TCR signaling pathway including the phosphorylation of Erk. pErk translocates into the nucleus and enhances the effector functional readouts of T-cell activation

Article Snippet: Anti-pLAT (Tyr191) (Cat# 3584S), P-p44/42 MAPK (T202/Y204) (Cat# 4370L), pSrc family (pY416) (Cat# 6943T), non-pSrc family (pY416) (Cat# 2102S), pSHP-1 (Cat# 8849S), pZap70 (pY319) (Cat# 2717S), Zap70 (Cat# 2709), pPLC-γ1 (pY783) (Cat# 2821S), α-tubulin (Cat# 3873S), GAPDH (Cat# 2118S) antibodies were from Cell Signaling Technologies.

Techniques: Phospho-proteomics, Functional Assay, Activation Assay