Figure 6. The Sema7a^R145W mutation does not alter total Sema7a expression but increases Sema7a and integrin β1 in the cell surface membrane and activates PKC-α signaling in hepatocytes.

Relative levels of Sema7a mRNA transcripts (A) and protein expression (B) in 10-week-old male WT, Sema7a^R145W heterozygous, and homozygous mice. One-way ANOVA with post hoc analysis. (C) Immunofluorescence and (D) IHC analyses of Sema7a protein in the livers of WT and Sema7a^R145W homozygous mice. Normal rabbit IgG was used as the negative control. Original magnification, ×200. (E) Relative expression levels of Sema7a and its receptors integrin β1 and plexin C1 in whole-cell lysates (left) and membrane fractions (right) extracted from primary mouse hepatocytes. (F) N-glycosylated-Sema7a protein (~130 kDa) was detected in primary mouse hepatocytes. Western blot revealed the deglycosylated Sema7a (black arrow). (G) Relative levels of Sema7a and integrin β1 proteins in whole-cell lysates (left) and membrane fractions (right) from human hepatoma HepG2 cells after transfection with the plasmid for SEMA7A_WT or SEMA7A_R148W. (H) Co-immunoprecipitation analysis of protein interactions among Sema7a, PKCα, and integrin β1 in liver tissues from 10-week-old male WT and Sema7a^R145W homozygous mice. (I) Phosphorylated PKC-α (T638) and PKC-α protein levels in whole-cell lysates (left panel) and membrane fractions (right panel) from primary mouse hepatocytes. T638 and PKC-α protein levels in (J) HepG2 cells that were transfected with SEMA7A_WT or SEMA7A_R148W plasmid and (K) primary mouse hepatocytes following integrin β1 silencing. (L) Co-immunoprecipitation analysis of protein interaction between Sema7a and PKC-α in primary mouse hepatocytes after integrin β1 silencing. Data are representative images or expressed as the mean ± SD of each group from 3 separate experiments. The difference among the groups was determined by 1-way ANOVA with Tukey’s post hoc tests or by Kruskal-Wallis test with Dunn’s post hoc test analysis, and the difference between the groups was analyzed by Student’s t test. *P < 0.05 versus WT mice (cells); ^#P < 0.05 versus Sema7a^R145W heterozygous mice (cells).