Figure 4: ERK1/2 signaling is involved in annexin A5-stimulated testosterone secretion in Leydig cells. ( a ) After incubation with annexin A5 (1 nmol l−1) for different time points, activated ERK (P-ERK) and total ERK (T-ERK) were detected by Western blotting. The ratio of P-ERK to T-ERK was calculated. The P-ERK level in Leydig cells without annexin A5 treatment (0 min) was set as a control. ( b ) Leydig cells were pretreated with 50 μmol l−1 PD98059 (ERK inhibitor) for 20 min, followed by treatment with 1 nmol l−1 annexin A5 for 24 h. The control culture was treated with Tris-HCl. Culture medium was collected for testosterone assay by chemiluminescence assay. Values are represented as mean ± s.d. from six individual experiments. *P < 0.05, **P < 0.01, compared with the control.