Figure 5: PKCζ promotes nuclear accumulation of TβRI.
From: TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer
(a) Immunofluorescence of endogenous TβRI visualized with the V22 antibody in PC-3U cells treated with TGFβ with or without PKCζ pseudosubstrate to inhibit PKCζ. Scale bar 20 μm. (b) Cell lysates from PC-3U cells treated with TGFβ or TPA were subjected to immunoblotting with pPKCζ antibody. Total cell lysates from cells transiently transfected with PKCζ siRNA served as negative control and cells treated with 10% FBS as positive control. The filter was reprobed with PKCζ antiserum to show equal loading of proteins in all lanes. (c) Cell lysates from wt and TRAF6−/− MEFs was subjected to immunoblotting for p-PKCζ,/PKCζ, TRAF6 and β-actin antibodies to show activation of PKCζ, knock down of TRAF6, and equal loading of proteins in all lanes, respectively. (d) PC-3U cells, in which endogenous PKCζ was silenced by its siRNA or not, and then treated with TGFβ, were subjected to cell fractionation followed by SDS–gel electrophoresis and immunoblotting to investigate the subcellular localization of endogenous TβRI. Lamin A and β-tubulin served as controls for the nuclear and cytoplasmic fractions, respectively. (e) Cell lysates from PC-3U cells transiently transfected and treated as indicated, in the presence or absence of wt PKCζ, subjected to immunoblotting to visualize TβRI-FL and TβRI-ICD. Immunoblotting of cell lysates for PKCζ and β-actin served as controls for the experiment. (f) Cell lysates from PC-3U cells transiently transfected with C-terminally HA-tagged wt TβRI and wt PKCζ, and treated with TGFβ in the presence and absence of TβRI inhibitor (SB505124) , subjected to immunoblotting for HA to visualize TβRI-FL and TβRI-ICD. Immunoblotting of cell lysates for PKCζ, p-Smad2 and β-actin served as controls for the experiment. (g) PC-3U cells were treated with TGFβ, with or without the PKCζ pseudosubstrate. Endogenous TACE and TβRI were visualized by immunofluorescence using TACE (TRITC) and V22 (FITC) antisera. Their colocalization is demonstrated by the yellow colour as shown in merge. Scale bar 20 μm. Stainings with DAPI was used to visualize cell nuclei in a, g.
