Gapdh clone 6c5

Western blotting was performed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system as previously described [9 (link), 10 (link)]. Immunoblotting was performed following incubation of samples with CUL4A (clone EPR3198, 1:10000, Abcam, Cambridge, MA), LC3B (catalog no. NB100–2220, polyclonal, 1:2000, Novus Biologicals, Littleton, CO), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; clone 6C5, 1:10000, Millipore, Burlington, MA) antibodies at 25°C for 2 h. The blots were washed and incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson, West Grove, PA, United States), followed by three washes with TBST (Tris-buffered saline + Tween). Enhanced chemiluminescent HRP substrate (Pierce, Rockford, IL, United States) was used for detection according to manufacturer’s description.

Briefly, approximately 35 µg of protein was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF, Biorad, USA). The membranes were blocked with skimmed milk (Merck, USA) and polyclonal antiPLCζ antibody (1:32000, Covalab, France), polyclonal anti-PAWP antibody (1:5000, abcam, UK) and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), clone 6C5 (1:5000, Millipore, USA), were used as specific primary antibodies. After three times washing, the secondary antibodies, used for PAWP and PLCζ, were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and for GAPDH was anti-rabbit IgG (all purchased from Dako, Japan). After three times washing, target proteins band were detected with an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, Germany). The fire reader (Uvitec, UK) was used for recording chemiluminescence images. Densitometric analysis of the images was performed by Quantity One Software v 4.6.9 (Bio-Rad, Germany). Results were expressed as mean relative intensity (mean intensity of the patient’s band/mean intensity of fertile bands) (17 (link)). Figure 1 showed Western blot of PLCζ and PAWP in infertile men with globozoospermia (n=4) and fertile men (n=5).

Total AQP2 was detected with antibodies (Pre‐C‐tail Ab) against the 20‐amino acid residue segment just N‐terminal from the polyphosphorylated region of rat AQP2 (CLKGLEPDTDWEEREVRRRQ) 12, 13. Alternatively, AQP2 was detected with a specific antibody (C‐tail Ab) raised against a synthetic peptide corresponding to the last 15 C‐terminal amino acids of human AQP2 14. AQP2‐pS256 antibodies were kindly gifted by Peter Deen and described by Trimpert et al. 15. Monoclonal antibody against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, clone 6C5) was purchased from Millipore (Millipore Corporation). Secondary goat anti‐rabbit and antimouse‐Alexa488‐conjugated antibodies were from Molecular Probes (Eugene, OR, USA).

Phenylephrine (SIGMA), MG132 (SIGMA). Sarcomeric Alpha Actinin (EA-53, Abcam), Cardiac Troponin I (Abcam), Gapdh (clone 6C5, Millipore), Myc (D84C12, CST), RNA polymerase II (8WG16, Millipore), Btg2 (custom-made polyclonal antibody by immunizing rabbits, Cyclex), Cnot7 (18W, Santa-Cruz), α-Tubulin (MBL), anti-FLAG (M2, SIGMA), and Alexa 488-and Alexa 568-labeled secondary antibodies (Invitrogen).