Activation of PKCη is necessary and sufficient for κ chain induction in the pre-B-cell lines. (A) BKO84 cells were cultured for 2 days with PMA, bisindolylmaleimide (Bis), Go6976, alone or in combination, or solvent alone (-), as indicated, and analyzed for κ chain expression by flow cytometry. (B) Reverse transcriptase-PCR analysis of PKCη and HPRT mRNA expression in BKO84, DKO35, 18-81, and WEHI231 cells. (C) BKO84 cells 4 days after infection with the indicated retroviral vectors were analyzed by flow cytometry. The bar graph indicates the frequency of κ-positive cells among GFP-positive (▪) or GFP-negative (□) cells. (D) BKO/ERtm and BKO/BASH-ERtm cells were treated with 1 μM 4-OHT (+) or solvent alone (-) for 4 days and analyzed by flow cytometry for the expression of κ chain. Numbers indicate percentages of the total live cells gated by light scatters. (E) Plasma membrane translocation of endogenous PKCη by BASH reconstitution. BKO/ERtm and BKO/BASH-ERtm cells were treated with 4-OHT (1 μM) for 1 hour, stained for surface pre-BCR, fixed and permeabilized, and stained for PKCη as described in “Materials and methods.” Images were obtained by confocal laser scanning microscopy and digitally colored in green (pre-BCR) and red (PKCη) and merged (Merge).