PKCε levels are modulated during MK differentiation. (A) Western blot detection of total PKCε protein expression in mouse MK cultures. GAPDH was monitored for protein loading. FLC, fetal liver cells; rMK, round MK; ppfMK, proplatelet-forming MK; PPL, proplatelets. (B) Relative PKCε protein expression during megakaryocytic differentiation from mouse FLC. Densitometric measurements of western blots from 5 replicates were performed by ImageJ software (means ± standard deviations [SD]; *P < .01 vs FLC culture day 0; #P < .05 vs rMK culture day 2). (C) Immunofluorescence analysis of PKCε and α/β-Tubulin expression and their colocalization in proPLT. Samples were examined with a confocal microscope Olympus FluoView FV10i (Center Valley, PA), with a 60× water-corrected objective. Each image represents a projection from a z-stack of 7 images collected at 0.5-µm increments. Images were obtained at 22°C and analyzed by ImageJ software. PKCε was detected with a rabbit polyclonal antibody and a secondary goat anti-rabbit antibody, conjugated to an Alexa Fluor 488; α and β tubulin were detected with mouse monoclonal antibodies and a secondary goat anti-mouse antibody, conjugated to an Alexa Fluor 568; MK nucleus was detected with DAPI.