Gapdh hrp

Cells were lysed in cold RIPA buffer supplemented with 1× protease inhibitor cocktail (Roche). Lysates were kept on ice for 30 min with vortexing every 5 min and then cleared by centrifugation (25,000 × g for 20 min at 4°C). Supernatants were collected, and protein contents were quantified by a BCA protein quantification assay (Pierce). After SDS-PAGE and transfer to nitrocellulose or polyvinylidene fluoride, membranes were blocked in 5% milk in 1× PBS and 0.1% Tween-20. For anti-biotin, blocking was performed in casein. In general, primary antibodies were applied overnight at 4°C, and secondary antibodies were applied for 1 hr at room temperature. Antibodies used included CCP110 (Proteintech; 1:1,000), CEP97 (Proteintech; 1:1,000), Biotin (Biotin-HRP Cell Signaling; 1:2,000), HA (Sigma; 1:1,000), GFP (Roche; 1:1,000), tubulin-HRP (Proteintech; 1:2,000), GAPDH-HRP (Proteintech; 1:1,000), and Actin (Sigma; 1:1,000). The anti-SALL1 antibodies used detect specifically SALL1^FL (R&D; aa 258–499) or the N-terminal part of SALL1.⁶ (link) Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit (Jackson Immunoresearch). Proteins were detected with Clarity ECL (BioRad) or Super Signal West Femto (Pierce). Quantification of bands was performed with ImageJ software and normalized against Actin or GAPDH levels. At least three independent blots were quantified per experiment.