Fig. 3

Effects of GDF9 variants on gene expression and GDF9-BMP15 interactions. A Western blots of mature GDF9 proteins expressed in follicular fluid collected from the first and second retrieval cycle of individual P1 and age-matched controls (n=3). B ELISA of GDF9 in follicular fluid collected from the first retrieval cycle of individual P1 and age-matched controls (n=6). C Western blots of GDF9 expression in HEK293T cells transfected with human GDF9^WT, GDF9^Q321X, GDF9^S428T, and GDF9^{His209GlnfsTer6} respectively and their relative mature-GDF9 expression in culture medium (CM) (3 independent experiments). The amount of concentrated culture medium used for the detection was 35μl per sample per assay. lnfsTer6, His209GlnfsTer6. *P < 0.05. D Western blots of GDF9 precursors and mature GDF9 over time during in vitro cleavage assay (3 independent experiments). Total GDF9=GDF9 precursor+mature GDF9. E Western blots of BMP15 expression in HEK293T cells co-transfected with human BMP15 and human GDF9^WT, GDF9^Q321X, GDF9^S428T, and GDF9^{His209GlnfsTer6} respectively and their relative BMP15 precursor expression in CM (3 independent experiments). lnfsTer6, His209GlnfsTer6. *P < 0.05. F Co-IP assay of the binding of GDF9 proteins to BMP15 protein. lnfsTer6, His209GlnfsTer6. *P < 0.05. G Western blots of p-smad2 and smad2 expression in human primary luteinized granulosa cells treated with purified recombinant pro-GDF9^WT-BMP15 and pro-GDF9^S428T-BMP15 protein respectively (3 independent experiments)