Journal:

Article Title: Tissue Hyperplasia and Enhanced T-Cell Signalling via ZAP-70 in c-Cbl-Deficient Mice

doi:

Figure Lengend Snippet: (A) Anti-CD3 stimulation of thymocytes from c-Cbl-deficient mice induces the tyrosine phosphorylation of proteins that are not detectably phosphorylated in normal thymocytes. Thymocytes from c-Cbl+/+, c-Cbl+/−, and c-Cbl−/− mice were left unstimulated or stimulated with anti-CD3 (αCD3) antibodies for 5 min at 37°C before lysis and immunoblotting with anti-phosphotyrosine (Anti-P-Tyr) antibodies. The 76-kDa phosphoprotein from the stimulated c-Cbl−/− thymocytes has a mobility equivalent to that of SLP-76. (B) Anti-CD3-induced phosphorylation of ZAP-70 in c-Cbl-deficient thymocytes is not dependent on Lck activation. Thymocytes from c-Cbl+/+ or c-Cbl−/− mice were left unstimulated (unstim) or stimulated with anti-CD3 or anti-CD3-CD4 antibodies for 5 min at 37°C. Cell lysates were either analyzed by immunoblotting with anti-phosphotyrosine antibodies or immunoprecipitated (i.p.) with anti-Lck or anti-ZAP-70 antibodies. The anti-Lck immunoprecipitates were analyzed in an immune complex kinase assay for Lck kinase activity, and the anti-ZAP-70 immunoprecipitates were analyzed by anti-phosphotyrosine immunoblotting.

Article Snippet: Anti-Lck antibodies were purchased from Zymed, anti-SLP-76 and anti-Fyn antibodies were purchased from Santa Cruz, anti-ZAP-70 antibodies were purchased from Transduction Laboratories and L. Samelson, and anti-phosphotyrosine (4G10) antibodies were purchased from B. Druker.

Techniques: Lysis, Western Blot, Activation Assay, Immunoprecipitation, Immune Complex Kinase Assay, Activity Assay

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A) JLAT transductants were stimulated with 5 μg/mL αCD3/CD28 for 15 min. mCherry coIP western blot shows that phosphorylation of LAT by DNA-PKcs at S241 is required for signalosome formation, given altered pull-down of GADS and SLP-76 in JLAT cells expressing S241A LAT and S2A LAT. Representative blots from n = 3 independent experiments. (B) CoIP coupled mass spectrometry was completed in JLAT transductants expressing WT, S224A, or S241A LAT stimulated with 5 μg/mL αCD3/CD28 for 15 min. Pull-down was normalized to mCherry-expressing JLAT cells, and protein pull-down was further normalized to relative LAT pull-down with log-fold differences shown between the groups, n = 5 replicates/transductant. The top statistically different proteins pulled down with mCherry-tagged LAT are represented in WT, S224A, and S241A LAT transductants.

Article Snippet: SLP-76 (D1R1A) Rabbit Monoclonal Antibody #70896 , Cell Signaling Technologies , Cat#70896; RRID: AB_2799792.

Techniques: Western Blot, Phospho-proteomics, Expressing, Mass Spectrometry