Mouse anti myc sc 40

Mouse anti-Myc (sc-40) and rabbit anti-paxillin (sc-5574) were from Santa Cruz Biotechnology. Mouse anti-pY31 paxillin was from BD Biosciences. Alexa Fluor 647 conjugated anti-human CD4 antibody was from SouthernBiotech. IRDye680CW goat antimouse IgG, and IRDye800CW goat anti-rabbit IgG were from Li-Cor Biotechnology. HEK293 cells and Jurkat cells were obtained from ATCC. The derivation and characterization of the HEK293-based Hck-expressing HZ-1 cells have been described elsewhere [19] (link). HEK293 and HZ1 cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS), 0.05 mg/ml penicillin, and 0.05mg/ml streptomycin. Lentiviral transduction was employed to generate Jurkat cells stably expressing human CD4. Briefly, HEK293 cells were co-transfected with 2.5 !g pDelta8.9, 1.5 !g VSV-G and 3 !g pWPI-puro plasmid containing human CD4 cDNA in Opti-MEM medium with 12 !g PEI. After 5 hours, medium was refreshed with cell culture media. Supernatant was collected 48 hours post-transfection, filtered and used to infect Jurkat cells. Infected cells were selected with 6 !g/ml puromycin for 2 days. Single cell clones of CD4 transduced Jurkat cells were isolated by cellenONE X1 system. Jurkat cells were maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS, 0.05 mg/ml penicillin, and 0.05 mg/ml streptomycin.