Figure 2

PKCθ-dependent Numb phosphorylation is regulated by pre-TCR. (a) Co-transfection of HEK293 cells with Flag-Numb p66 with or without CA-PKCθ expression plasmids. Whole-cell lysates were subjected to immunoprecipitation using anti-flag antibody and were revealed in western blot with anti-P-ser PKC substrate antibody. All samples were supplemented with empty vector so that the final concentration of DNA was the same in all reactions. (b, left panel) Total cell lysates of SCB29 and SCIET27 cells were resolved by western blot and incubated with anti-P-PKCθ (Thr538) and anti-β-actin antibodies. (Right panel) Endogenous Numb immunoprecipitates in whole-cell lysates of SCB29 and SCIET27 cells were incubated with anti-P-ser PKC substrate and anti-numb antibodies to confirm that equal Numb amounts were immunoprecipitated. (c, right panel) Numb immunoprecipitates in total lysates of non-treated or treated SCB29 cells with 3 uM of Rottlerin for 1 h were incubated with anti-P-ser PKC substrate and anti-numb antibodies. (Left panel) Total cell lysates from the same experiment were incubated with anti-P-PKCθ (Thr538) and anti-β-actin antibodies. (d and e) Numb immunoprecipitates in whole-cell lysates of (d) SCIET27 cells or (e) thymocytes from pTα^−/−, treated or non-treated with 5 ng/ml of PMA for 1 h, were incubated with anti-P-ser PKC substrate and anti-numb antibodies. All samples were supplemented with empty vector so that the final concentration of DNA was the same in all reactions. All data are representative of three independent experiments