Figure 3: Knockdown of annexin A5 inhibits testosterone production and the expression of testosterone synthesis-related enzymes in rat Leydig cells. ( a ) Leydig cells were transfected with three different siRNAs targeting annexin A5, and the protein expression of annexin A5 was detected at 48 h after transfection by Western blotting. ( b ) Leydig cells were transfected with annexin A5 siRNA for 48 h, then culture medium was collected and assayed for testosterone production by chemiluminescence assay. ( c ) Protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD in Leydig cells was detected after annexin A5 knockdown. The densitometric analysis of Western blots was determined by the normalization to internal control β-actin. Values are shown as mean ± s.d. from at least three independent experiments. *P < 0.05, **P < 0.01, compared with negative control.