Fig. 5

PDIA3P1 stabilizes C/EBPβ by preventing MDM2 -mediated ubiquitination. A Different protein bands pulled down by PDIA3P1 junction sense or anti-sense in GSC267 cells. B RNA pull down assay showing the interaction between C/EBPβ with PDIA3P1. C RIP and qRT-PCR assays revealing the interaction between C/EBPβ with PDIA3P1. D The protein expression effected by PDIA3P1 knockdown and C/EBPβ overexpression. E F Overexpression of C/EBPβ rescued the effect of PDIA3P1 knockdown on self-renewal ability of GSC267. Scale bar, 200 μm. G H Knockdown of C/EBPβ inhibited the effect of PDIA3P1 overexpression on self-renewal ability of GSC8–11. Scale bar, 200 μm. I Quantification of comet assay and γ-H2AX staining of GSC267 under TMZ treatment (400 μM, 48 h). J Quantification of comet assay and γ-H2AX staining of GSC8–11 under TMZ treatment (400 μM, 48 h). K Western blotting analysis of the effect of PDIA3P1 knockdown on C/EBPβ with or without MG132 treatment (10 μM, 12 h). L Western blotting analysis of C/EBPβ in PDIA3P1 stable knockdown and control GSC267 cells after treatment with CHX (100 μg/ml) for indicated times. M GSCs lysates were immunoprecipitated with anti-C/EBPβ antibody followed by immunoblotting with anti-Ubiquitin antibody and anti-C/EBPβ antibody. The GSCs were pretreated with MG132 (10 μM) for 6 hours. N Co-IP analysis of interaction between C/EBPβ and MDM2 in GSC267 cells transfected with PDIA3P1 or shPDIA3P1