Anti phospho plcγ1 tyr783

Mouse CD4⁺ T cells from WT C57BL/6 or TRAIL-R-KO mice were enriched (STEMCELL Technologies) and stimulated at the indicated time point at 37°C with medium, an anti-CD3 Ab (1 µg/ml), an anti-CD28 Ab (1 µg/ml), and the TRAIL (10 µg/ml), or a combination of anti-CD3/anti-CD28 Abs and the TRAIL. Whole-cell protein was subsequently extracted using the PhosphoSafe Extraction Reagent (Merck Millipore, Darmstadt, Germany). Protein lysates were transferred onto a polyvinylidene difluoride membrane after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and labeled using monoclonal primary Abs against anti-phospho-ZAP70 (Tyr319) (Cell Signaling, Beverly, MA, USA), anti-ZAP70 (Cell Signaling), anti-phospho-LAT (Tyr191) (Abcam, Cambridge, UK), anti-LAT (Thermo Scientific), anti-phospho-PLCγ1 (Tyr783) (Cell Signaling), anti-PLCγ1 (Abcam), and anti-β-actin (Abcam) Abs. The secondary Ab was labeled with HRP, and electrochemiluminescence was used to visualize the bands.

Anti-PDGFRβ (958) and anti-phosphotyrosine (PY99) antibodies were purchased from Santa Cruz Biotechnology, Dallas, Texas, USA. The anti-PDGFRβ (AH 17.2) monoclonal antibody was produced as described [24 (link)]. Anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PLCγ1 (Tyr783) and anti-PLCγ antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA. Anti-calnexin antibody was purchased from Enzo® Life Sciences, Farmingdale, NY, USA. Secondary antibodies used in western blot experiments were purchased from Cell Signaling Technology. The secondary antibody coupled to phycoerythrin used in flow cytometry was purchased from Jackson ImmunoResearch, West Grove, PA, USA. Fluorescent secondary antibodies were purchased from LI-COR Biosciences, Lincoln, NE, USA. PDGF-BB was purchased from PeproTech, Rocky Hill, NJ, USA. Imatinib was purchased from LC Laboratories (Woburn, MA, USA). Cycloheximide was purchased from Sigma. ATP was purchased from Fermentas (Thermo Fisher Scientific, Waltham, MA, USA). EZ-Link Sulfo-NHS-Biotin and streptavidin agarose beads were purchased from Pierce (Thermo Scientific).

Protein G-coated dynabeads were obtained from Novex Life technologies. Pan T cell isolation kit II was from Miltenyi Biotec. HIT3a anti-CD3 mAb and CD28.2 anti-CD28 mAb were from Becton Dickinson. Anti- LAP-TGFβ mAb; latent TGFβ1,; active TGFβ1 cytokine, cytokine array kit and TGFβ cytokine ELISA assay were from R&D Systems. Anti-phospho-PLCγ1 (Tyr783), anti-phospho-Zap-70 (Tyr319), and anti-phospho-LAT (Tyr191) were obtained from Cell Signaling Technology, anti-β-Actin monoclonal antibody from Sigma. HRP-conjugated donkey anti-mouse IgG or donkey anti -rabbit IgG (Dianova, Hamburg, Germany) were used as secondary antibodies. Ficoll was from Biochrome, [³H]thymidine from MP Biomedicals, AIM-V culture medium from Invitrogen.