Fig. 4

Effects of p-KAP1 on the stability of PCNA in NSCs undergoing cerebral I/R. A Real-time PCR analysis of PCNA mRNA levels in C17.2-NSCs after OGD/R. Data are the mean values ± SEM of three replicate experiments, NS indicates no significance. B Western blot analysis of PCNA protein levels in C17.2-NSCs after OGD/R. C17.2-NSCs were pre-incubated with 20 μM MG132 for 6 h before harvesting cells for protein lysis. C Band intensities are represented as fold-values relative to the levels in the normoxia group. Data were expressed as mean ± SEM (n = 4). ^##P < 0.01 versus DMSO and O/R group. D C17.2-NSCs were treated with 20 μM MG132 for 6 h before harvesting for protein lysis and PCNA was precipitated with the anti-PCNA antibody. The protein levels and ubiquitination levels of PCNA in the immune complex were determined by Western blot. The protein levels of PCNA and α-Tubulin in cell lysates (input) were also analyzed by Western blot, and three independent experiments were performed. “a” indicates the heavy chain of IgG, “b” indicates the light chain of IgG. E PCNA and CUL4A colocalization immunofluorescence staining was performed to evaluate the neurogenesis of endogenous NSCs. 200× , scale bar = 100 μm