Association of protein kinase Cδ phosphorylation/activation with PLSCR1 expression. (A) After treatment with 10^-6 M ATRA, 100 nM PMA, 1% DMSO, or 2.5 × 10^-7 M VD3 for the times indicated, NB4 cells were extracted by phosphorylation lysis buffer for Western blots. The blots were probed with antiphospho-PKC-δ (Ser643); then the blots were stripped and reprobed with antibody against PKC-δ. (B, top) After preincubation for 2 hours in the presence or absence of 4 μM rottlerin, NB4 cells were treated with 10^-6 M ATRA or 100 nM PMA for 8 hours. After lysis with phosphorylation lysis buffer, equal amounts of total cell lysates were analyzed by Western blot with antiphospho-PKC-δ (Ser643); then the blots were stripped and reprobed with antibody against PKC-δ. (B, bottom) After preincubation for 2 hours in the presence or absence of 1 μM rottlerin, NB4 cells were treated with 10^-6 M ATRA or 100 nM PMA for 3 days. After lysis with ice-cold lysis buffer (see “Materials and methods”), equal amounts of total cell lysates were analyzed by Western blot for PLSCR1. In all cases, β-actin served as loading control. Similar results were observed for U937 cells (data not shown). (C) Cos-7 cells were transiently transfected without (CON) or with 1.5 μg empty vector pEGFP-N1 (N1), or 1 μg (CF1, supplemented by 0.5 μg empty vector) and 1.5 μg (CF1.5) pEGFP-CF-PKCδ containing the catalytic fragment of PKCδ. At 48 hours after transfection, semiquantitative RT-PCR (top and middle) and Western blot (bottom) for PLSCR1 were performed.