Fig. 4

SSFA2 interacted with GSTM3 and Actin during spermatogenesis. A In a healthy control, Actin was located in the head and tail, and SSFA2 was located in the acrosome. Actin and SSFA2 signals were barely detectable in the sperm head of the patient (green, SSFA2; red, Actin; blue, DAPI; scale bars, 1 μm). B The interaction between SSFA2 and Actin was identified by coimmunoprecipitation. C Colocalization of SSFA2 and Actin was examined by immunofluorescence in testes from healthy controls (green, SSFA2; red, Actin; blue, DAPI; scale bars, 100 μm). D In spermatozoa from a healthy control, GSTM3 localized to the tail and equatorial plate and colocalized with SSFA2 on the equatorial plate, but only GSTM3 localized to the flagella was detected in the patient sperm (green, SSFA2; red, GSTM3; blue, DAPI; scale bars, 1 μm). E The interaction between SSFA2 and GSTM3 was identified by coimmunoprecipitation. F Colocalization of SSFA2 and GSTM3 was examined by immunofluorescence in testes from healthy controls (green, SSFA2; red, GSTM3; blue, DAPI; scale bars, 100 μm). G, H In vitro experiments demonstrated the interaction between SSFA2 and GSTM3. The Myc-WT-GSTM3 plasmid and the Flag-WT-SSFA2 plasmid were cotransfected into the HEK293T cell line, and immunofluorescence revealed that GSTM3 and SSFA2 colocalized in the cytoplasm. Further co-IP analysis also verified their interaction (green, SSFA2; red, GSTM3; blue, DAPI; scale bars, 100 μm)