PKCε expression is necessary for proPLT formation. (A) Western blot detection of PKCε, PKCδ, PKC\x{03b8}θ, and PKCα in mouse MK untreated (UN) or infected with PKCε-specific shRNAs (shRNA59, shRNA60) and nontarget shRNACT. GAPDH was monitored for protein loading. (B) Densitometric analysis of PKC protein expression was performed using ImageJ software. Densitometric measurements of western blots from 3 replicates (means ± 1 SD; *P < .05 vs shRNACT ANOVA and Tukey tests). (C) Representative images of proplatelet-forming MK infected with PKCε-specific shRNAs (shRNA59, shRNA60) or control (shRNACT), isolated at day 4 by gradient and cultured for an additional 24 hours. (D) Immunofluorescence analyses of proplatelets from MK infected with PKCε-specific shRNA (shRNA59, shRNA60) and control (shRNACT), isolated at day 4 by gradient separation and cultured for a further 24 hours. Samples were labeled with specific antibodies against α/β-Tubulin and PKCε. The composite panels show the colocalization of PKCε and α/β-Tubulin at the tubular coil (particularly in released platelets, as shown in the insert) in shRNACT, and the abnormal morphology, in terms of dimension and tubular coil definition, of platelets released by PKCε-specific shRNA infected MKs. Samples were examined with microscope Axiovert 200 (Carl Zeiss, Inc., Thornwood, NY), equipped with a 63× numerical aperture 1.4 oil immersion objective. Images were obtained using a CCD camera and analyzed using the MetaMorph image analysis software. Images were obtained at 22°C and analyzed using 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 rmouse monoclonal antibodies and a secondary goat anti-mouse antibody, conjugated to an Alexa Fluor 568.