Anti zap 70

Jurkat cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 50 U ml⁻¹ penicillin and 50 μg ml⁻¹ streptomycin at 37 °C in an atmosphere of 5% CO₂/95% air. Jurkat T cells were infected with control shRNA lentiviral particles (sc-108080, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or CHMP5 shRNA (h) lentiviral particles (sc-60374-V, Santa Cruz) and selected based on the manufacturer's protocols. Control (Ctrl) Jurkat and CHMP5^KD Jurkat cells were maintained and grown in RPMI 1640 media supplemented with 10% fetal bovine serum (Sigma-Aldrich), 50 U ml⁻¹ penicillin, 4–8 μg ml⁻¹ puromycin and 50 μg ml⁻¹ streptomycin at 37 °C in an atmosphere of 5% CO₂/95% air. The antibodies used were anti-CHMP5 (Abcam, Cambridge, CO, USA), anti-GAPDH (Santa Cruz Biotechnology), anti-TCRαβ (BD Biosciences, San Jose, CA, USA), anti-CD3 (BioLegend, San Diego, CA, USA), anti-CD28 (BioLegend), anti-TCRβ (Abcam), anti-pho-PKCθ (Cell Signaling Technology, Danvers, MA, USA), anti-PKCθ (Cell Signaling Technology), anti-pho-IKKαβ (Cell Signaling Technology), anti-IKKα (Cell Signaling Technology), anti-pho-ZAP-70 (Santa Cruz Biotechnology), anti-ZAP-70 (Santa Cruz Biotechnology), anti-pho-Lck (Santa Cruz Biotechnology) and anti-Lck (Santa Cruz Biotechnology).