Horseradish peroxidase conjugated anti rabbit and anti mouse iggs

Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich Quimica S.A. (Madrid, Spain). Sydney IVF Sperm Medium was provided by Cook Medical (Barcelona, Spain). 4–15% polyacrylamide gels were supplied by Bio-Rad (Madrid, Spain) and PVDF membranes by Merck (Madrid, Spain). The primary antibodies were: anti-phospho-PKA substrates (PKAs-P) (Cell Signaling Technology, Beverly, USA, #9624), anti-phosphotyrosine (Tyr-P) (Abcam, Cambridge, UK, #ab10321) and anti-β-tubulin (β-TUB) (Sigma-Aldrich Quimica S.A., Madrid, Spain, #T0198)^{78 (link),79 (link)}. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were obtained from Santa Cruz Biotechnology (Heidelberg, Germany, #sc2004) and Bio-Rad (Madrid, Spain, #1706516), respectively. PageBlue was provided by Thermo Scientific (Rockford, IL, USA), whereas acetonitrile, trifluoroacetic acid and formic acid by Fisher Scientific (UK). Trypsin Gold Proteomics Grade and ProteaseMax surfactant were purchased from Promega Corporation (Madison, MI, USA).

Protein isolation from cultured fibroblasts of individual III-1 from family A and Western blot were carried out as described [6 (link)]. The primary antibodies used for immunodetection were rabbit anti-WDR11 (Novus Biologicals, NBP1-89930, 1:500, N-terminal epitope aa 268-348) and rabbit anti-WDR11 (Abcam, ab93871, 1:500, C-terminal epitope aa 1174–1224). Rabbit anti-α-Tubulin (Abcam, ab15246) and mouse anti-beta-Actin (Abcam, ab6276) antibodies were used to control equal loading of protein extracts. As secondary antibodies, horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs (Santa Cruz Biotechnology, sc-2370/sc-2005) were used. PageRuler Prestained (ThermoScientific) and biotinylated Protein Ladders (Cell Signaling, 7727S) were used for protein molecular weight estimation. Detection was done using Clarity Western ECL Substrate (Bio-Rad) and the FujiFilm LAS 3000 system.