Fig 1: PKCa phosphorylates AKAP12 and inhibits its interaction with HSP47.(A) AKAP12 is phosphorylated by PKCa at its activation-responsive phospho-sites. Recombinant WT or AKAP12 phospho-mutants were in vitro translated from their vectors and subjected to in vitro kinase assay in the presence of active PKCa enzyme as in Materials and methods. The reactions were run on a phostag gel to detect phosphorylated AKAP12 or its mutants. Representative phostag gels from three experiments are shown. Source data are presented in Figure 3—source data 1. P values are calculated in Figure 3—source data 4. (B) Direct Interaction between AKAP12 and HSP47 in recombinant system in the absence or presence of active PKCa enzyme. In vitro translated biotinylated AKAP12 was incubated with recombinant HSP47 antibody column containing bound HSP47 (left) or recombinant HSP47 was incubated with Biotin antibody column containing bound AKAP12-Biotin (right) in the presence or absence of active PKCa as in Materials and methods. Two representative data out of four experiments are shown. Source data are presented in Figure 3—source data 2. (C) Silencing Prkca in activated human HSCs enhances AKAP12-HSP47 interaction. Culture-activated human HSCs were transfected with a universal negative control siRNA (Neg) or two Prkca siRNAs (A or B) as in Materials and methods. Total protein was assessed for AKAP12-HSP47 co-immunoprecipitation or PKCa, HSP47, and GAPDH immunoblotting. Data represented as GAPDH normalized densitometry are mean ± SE from five experiments. Source data are presented in Figure 3—source data 3. P values are calculated in Figure 3—source data 4. HSC, hepatic stellate cell; WT, wild-type. Figure 3—source data 1.Source blots for Figure 3A. Figure 3—source data 2.Source blots for Figure 3B. Figure 3—source data 3.Source blots for Figure 3C. Figure 3—source data 4.Post hoc analysis for Figure 3C.