Fig 2: Effect of phosphatase inhibitors on PP1, PP2A, and PP2B enzymatic activity.Sperm were selected and re-suspended in NCM. Each phosphatase activity was measured in sperm extracts as indicated in Materials and Methods in the absence of inhibitors (black bars) and then in the presence of 10 nM okadaic acid (white bar); 40 µg/ml I2 (grey bar); 90 nM endothall (dotted bar); 0.1 nM okadaic acid (light grey bar); or 0.1 nM deltamethrin (striped bar). Results were obtained from five different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p<0.05; ** p<0.005; *** p<0.001. The enzymatic activity is expressed as relative activity.

Fig 3: PP1-mediated PAR2 activation induced dephosphorylation of YAP. a Whole-cell lysate of HT-29 was immunoprecipitated with anti-YAP antibody or control IgG, followed by western blot for PP1, PP2a, PP2Ab, and PP2Ac. b HEK293 cells were treated with PAR2-AP for 30 min. Then, cell lysates were collected and used for immunoprecipitation with anti-YAP antibody. PP1 were detected by western blot. c Twenty-four hours after transfection with two different siRNAs against PP1, HEK293 cells were serum-starved for 12 h and then stimulated with PAR2-AP for 30 min. Western blot was used to determine p397-YAP and YAP. d The density of WB bands from panel d was analyzed by image-pro-plus soft system. Fold change of YAP phosphorylation is calculated as [p397-YAP /YAP]PAR2-AP/[p397-YAP /YAP]NC. Data were shown as mean ± SEM. *p < 0.05 vs. NC control. e Diagram of signaling pathways responsible for PAR2-regulated YAP stability and function

Fig 4: Presence of PP1?, PP2B, and PP2A in human sperm.Twenty-microgram samples of sperm extracts were subjected to SDS-PAGE and then transferred onto a PVDF membrane. The membrane was then probed with anti PP1? (PP1?), anti-PP2Ba (PP2B), and with an antibody sampler kit anti PP2A (PP2Ac, PP2A-A, PP2A-B). The western blot depicted is a representative experiment repeated at least three times. Molecular mass is indicated at the right.

Fig 5: Specific enzymatic activity of PP1 (A), PP2A (B), and PP2B (C) during human sperm capacitation.Sperm were selected and re-suspended in NCM and the PPs activity was measured immediately (black bar, T0). Then, sperm aliquots were resuspended in RCM and incubated at 37°C and 5% CO2 for up to 300 min. Different aliquots were taken at the indicated times and the PPs activity measured as indicated in Materials and Methods (white bars). Results were obtained from four different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p<0.05; ** p<0.005; *** p<0.001. The enzymatic activity is expressed as relative activity. In the insert, the upper panel represents the expression of PP1 (A), PP2Ac (B), and PP2B (C) during the corresponding capacitation times; the lower panel corresponds to the load control with anti ß-tubulin antibody.