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Proteins were subjected to 8% to 20% gradient gels of SDS‐PAGE (GE Healthcare) or 4% to 15% gradient gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. Membranes were blocked (PBS, 0.1% Tween 20, 5% bovine serum albumin) for 1 hour. Membranes were probed in blocking buffer containing primary antibodies of 1:1000 dilution: phosphorylated eNOS at serine 1177 (BD Biosciences), followed by the appropriate horseradish peroxidase–conjugated secondary antibody. Immunoreactions were visualized with Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare). Membranes were stripped (62.5 mmol/L Tris‐HCl [pH 6.8], 100 mmol/L mercaptoethanol, 2% SDS) for 30 minutes at 50°C and reprobed with O‐GlcNAc (RL2; Abcam), eNOS/NOS type III, antiactin, or glyceraldehyde‐3‐phosphate dehydrogenase antibodies of 1:1000 dilution to verify equal protein loading. The bands were quantified by densitometry (LAS‐3000 mini; FUJIFILM).

Antibodies were raised against PRR19 C-terminal fragment (183 amino acids between Thr183 and Tyr366 residues) and CNTD1 C-terminal fragment (124 amino acids between Cys126 and Thr249 residues). Coding sequences corresponding to these peptides were cloned into pDEST17 bacterial expression vector. Recombinant 6xHis-tagged proteins were expressed in E. coli strain BL21 tRNA and subsequently purified by Ni-Sepharose beads and SDS-PAGE (Amersham, GE Healthcare). Either elution fractions or homogenised SDS-PAGE gel fragments containing the purified proteins were used for immunisation of rabbits and guinea pigs. PRR19 or CNTD1 fragments coupled to NHS-activated Sepharose 4 Fast Flow beads (Amersham, GE Healthcare) were used to affinity purify polyclonal antibodies following standard procedures. Goat anti-RNF212 was raised against full-length mouse RNF212 protein. The specificity of anti-PRR19, anti-CNTD1 and anti-RNF212 antibodies was confirmed by: (1) immunoprecipitations and western blot analysis of protein extracts from testes of wild-type and Prr19^−/−, Cntd1^−/− and Rnf212^−/− mice, respectively; (2) immunostaining of spermatocyte nuclear surface spreads from testes of wild-type and mutant mice.