Quantifying Protein Expression Using Western Blot

To determine the efficiency of the shRNAs, we analysed the expression levels of the protein of interest in control and shRNA-treated samples, by SDS–polyacrylamide gel electrophoresis protein separation and immunoblotting. Protein extracts from P19 cells or whole-telencephalon tissue were obtained by lysis with the Promega passive lysis buffer complemented with a cocktail of protease inhibitors (Roche). Western blotting was performed using standard protocols55 (link). The primary antibodies were diluted in the blocking solution at the following concentrations: rabbit anti-Cenpj: 1:400 (Proteintech), rabbit anti-actin, 1:1,000 (Sigma, A2066) and rabbit anti-alpha tubulin, 1:1,000 (Sigma) and mouse anti-Mash1, 1:1,000, BD bioscience. Horseradish peroxidase-conjugated secondary antibodies were used as follows: goat anti-rabbit, 1:200 (Dako, P0449) and goat anti-mouse, 1:1,000 (Sigma, A8924). Uncropped scans of Fig. 1b and Supplementary Fig. 3f blots are shown in Supplementary Fig. 6.