PMA treatment reduced the tyrosine phosphorylation of CBL and dissociation of binding of CRKL to CBL. Immunoprecipitations of U937 cell lysates were performed with anti-CBL antibody after PMA or FcγRI stimulation. Lane 1 & 2 indicates preimmune and resting state (NS) respectively. Lane 3, 4 & 5, treated with PMA (200 ng/ml) for 1, 5 and 10 min. respectively. Lane 6, 7 & 8, stimulated for FcγR1 with 32.2 F(ab)2 and cross linked with secondary antibody (rabbit anti mouse F(ab’)2) for 1 , 5 and 10 min. respectively. Whole cell lysate was added in lane 9 as positive control (WCL). Immunoprecipitated proteins were resolved in 10% SDS-PAGE, blot on nitrocellulose membrane and probed with anti-phosphotyrosine antibody (4G10). This blot was again reprobed with anti-CBL and anti-CRKL antibody. This experiment was repeated three times.
Joshi et al. BMC Immunology 2014 15:18 doi:10.1186/1471-2172-15-18