Immunoblotting assays were performed by treating NB cells with different concentrations of CUDC-907 for 6 h, followed by cell lysis using RIPA buffer (89900; Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Complete mini EDTA free, Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (PhosSTOP, Roche). Protein samples were quantified with Bradford assay (5000205; Bio-Rad, Hercules, CA, USA) and equal amounts of total protein were separated on 4–12% SDS-PAGE gels, followed by transfer to PVDF membranes using a Bio-Rad Trans-Blot Turbo TM system, then blocking with 5% BSA solution, and probing with corresponding primary antibodies (1:1000 dilution) overnight at 4 °C [25 (link)]. This was followed by washing and incubating the membranes with either anti-mouse or anti-rabbit IgG HRP-conjugated secondary antibody (1:10,000 dilution) for 2 h. Protein bands on membranes were developed using Clarity ECL Western substrate (Bio-Rad) and visualized using the ChemiDoc XRS Plus system (Bio-Rad). Densitometric analysis of the protein bands was performed using the ImageJ software.
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