Fig. 7: PCSK9 phosphorylated Syk and PKCδ, and their phosphorylation was dependent on CAP1, not on LDLR.

a Immunoblot analysis of signal activation of Syk, PKCδ, AKT, and NF-κB p65 after rhPCSK9 (2 µg/mL) treatment in a time-dependent manner (0, 20, 40, 60, and 120 min) in the THP-1 cell line. Syk and PKCδ were phosphorylated after 20 min of rhPCSK9 (2 µg/mL) treatment. AKT phosphorylation started at 40 min and lasted until 60 min (N = 3). b, c Multiplex ELISA of cAMP secretion to assess the involvement of Syk, PKCδ, and PKA in the PCSK9-induced inflammation pathway (N = 4). b R406, rottlerin, and H892HCl were used to inhibit Syk, PKCδ, and PKA, respectively. Rottlerin blocked PCSK9-mediated cAMP induction, whereas R405 and H892HCl did not (N = 3). c, d Immunoblot analysis demonstrating that PCSK9-induced the phosphorylation of Syk, PKCδ, and AKT in THP-1 cells. The phosphorylation was attenuated in cells transfected with CAP1 siRNA (N = 4 for p-Syk and p-AKT; N = 3 for p-PKCδ). e Immunoblot analysis of Ldlr^−/− BMDMs, demonstrating that PCSK9-induced phosphorylation of Syk, PKCδ, AKT, and NF-κB p65 was independent of LDLR (N = 3). f Ldlr^−/− mice arteries were partially ligated and treated with AdV-CTRL or AdV-PCSK9, followed by a comparison of arteries exposed to S-flow and D-flow. Immunofluorescence staining for analyzing the expression of PCSK9 (red, upper panels; N = 3), p-Syk (gray, middle panels; N = 4), and p-PKCδ (green, bottom panels; N = 4). The expression of PCSK9 significantly increased in the group treated with AdV-PCSK9 compared with that in the control group. Additionally, p-Syk and p-PKCδ increased more significantly under D-flow of AdV-PCSK9-treated mice than in the control group. The scale bar represents 10 μm. The differences between the groups were compared using the unpaired t-test (two-tailed). All experiments are independently performed. Data are presented as mean values ± SD and SEM (in (f) only).