Fig. 5

Validation of SRD5A2 wild-type (WT) and p.H232R mutant HEK293 cell models. a. The HEK293 cells were transiently transfected with WT or p.H232R (c.695A > G) mutant SRD5A2 plasmids. Sequencing analysis showed the mutation c.695A > G in SRD5A2 in HEK-293 cells transfected with p.H232R mutant plasmids, causing amino acid 232 to change from histidine to arginine. b. The transcription of the SRD5A2 gene in HEK-293 cells transfected with WT and p.H232R mutant plasmids using qRT-PCR. The β-actin gene was used as an internal control. The amounts of SRD5A2 transcripts were calculated by the standard 2 − ΔΔCt method and were made into a histogram. c. Western blot results of SRD5A2 protein in HEK-293 cells transfected with WT-Flag and p.H232R-Flag mutant plasmids. β-Actin was used as an internal control. d. Enzyme activity analysis of SRD5A2 WT and mutants H232R binding with T by LC–MS. e. Enzyme activity analysis of SRD5A2 WT and mutants H232R binding with NADPH by LC–MS