Mouse anti cd3ζ 6b 10

Duolink™ (OLINK Bioscience) was performed according to the manufacturer’s instructions. Briefly, the cells were incubated for 30 min on IBItreat μ-channels (IBIDI) coated with poly-L-Lysine, fixed for 10 min in 4% paraformaldehyde prewarmed at 37 °C, permeabilized in PBS, 0.2% saponin for 10 min and blocked with 0.2% BSA in PBS, 0.1% saponin. Primary antibodies used were: mouse anti-CD3ε (OKT3, Biolegend), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), rabbit anti-IRAP (a generous gift from S. Keller, Virginia University, USA), mouse anti-Lck (3A5, Santa Cruz Biotechnology), rabbit anti-LAT (Millipore), rabbit anti-Rab7 (H-50, Santa Cruz Biotechnology) and rabbit anti-IRAP (D7C5), mouse anti-IRAP (3E1) and rabbit anti-ZAP-70 (D1C10E) (all from Cell Signaling Technology). All primary antibodies were diluted at 1/100, except mouse anti-CD3ζ (1/50). After washing the cells, PLA probes were added, followed by hybridisation, ligation, and amplification for 100 min at 37 °C. Protein interactions were visualised after incubation with the detection solution. Fluorescence signal was acquired on an LSM 510 Zeiss confocal microscope.

Abs used for Western blotting analysis were: mouse anti-Csk (BD Biosciences), rabbit anti-Csk (C-20), rabbit anti-TRAF3 (H122,), mouse anti-CD3_ζ (6B10.2, all from Santa Cruz), rabbit anti-pCsk S364 (Abcam), rabbit anti-PTPN22 (D6D1H), rabbit anti-pSrc 416, rabbit anti-Lck, rabbit anti-pLck 505, rabbit anti-LAT (all from Cell Signaling), mouse anti-FLAG (M2), mouse anti-HA (HA-7, all from Sigma), mouse anti-pY (4G10) and mouse anti-actin (Clone 4, Millipore). Western blot analysis was performed as in ref. 13 (link).