Transfection of PKCϵ and PKCϵm in erythroleukemic cell lines. (A) Detection of exogenous PKCϵ-GFP and endogenous wild-type PKCϵ protein by Western blot. Phosphorylated PKCϵ (pPKCϵ) was detected by specific C-terminal anti–phospho-Ser729 antibody. β-Actin was monitored for protein loading. (B) Cell-surface expression of TRAIL-Rs after PKCϵ transfection. TF-1 cells were transfected with PKCϵ-GFP (filled histograms) or PKCϵm-GFP (open histograms), and TRAIL-R expression was monitored by flow cytometry 48 hours later. (C) PKCϵ reduces the sensitivity of erythroleukemic cell lines to TRAIL-induced apoptosis. K562, HeL, and TF-1 cell lines were transfected with PKCϵ (▪) or with PKCϵm (□) and 48 hours later were treated with 50 ng/mL TRAIL. Residual cell viability was analyzed by flow cytometry, staining cells with annexin V–FITC and PI. The mean of 3 independent experiments is reported as percentage of control. Controls were represented by mock-transfected cell lines treated with TRAIL. *P < .05.
