Chemidoc xrs plus instrument

SDS-PAGE, immunoprecipitation, and western blot assays were carried out as described previously (Tanigawa et al. 2013 (link); Pan et al. 2010 (link)). In brief, cell lysates were centrifuged at 500 × g for 5 min at 4 °C to prepare nuclei and cytosol by using the nuclear and cytoplasmic extraction reagents, respectively (NE-PER; 78833; Thermo Fisher Scientific). Cell lysates were fractionated in 10% SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (0.45-μm IPVH00010; Merck) for 1 h at 100 V (fixed) at 10°C using a Mini Trans-Blot transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Blotted proteins were visualized with Ponceau S (Merck, P17170) to examine the amounts of the transferred proteins. PVDF membranes were then incubated with primary and secondary antibodies (Supplementary Table S1). Results were investigated using a ChemiDoc XRS Plus instrument (Bio-Rad). Immunoprecipitation was conducted by protein A/G beads coated by indicated antibodies (Tanigawa et al. 2013 (link); Pan et al. 2010 (link)).

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), IP, and Western blotting assays were performed as described previously [33 (link)]. Briefly, cells were lysed on ice with RIPA buffer (50 mM Tris–HCl, pH 7.4, with 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM ethylenediaminetetraacetic acid (EDTA)) containing protease and phosphatase inhibitors (1 mM phenylmethyl sulfonyl fluoride, 1 mM Na₃VO₄, 1 mM NaF, and 1 μg/mL of aprotinin, leupeptin, and pepstatin A each; all added immediately before use). In some experiments, cell lysates were centrifuged at 500 × g for 5 min at 4 °C to prepare nuclei and cytosol fractions using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific). All lysates were fractionated by SDS-PAGE and transferred to a 0.45 μm Immobilon®-P polyvinylidene difluoride (PVDF) membrane (Merck, Darmstadt, Germany; IPVH00010) for 1 h at 100 V (fixed) at 10 °C using a TE22 Mighty Small Transfer Tank system (BioCompare, San Francisco, CA, USA). Blots were stained with Ponceau S (Merck; P17170) to monitor the transferred protein amounts. PVDF membranes were then probed with the primary and secondary antibodies (Table S2). The results were analyzed using a ChemiDoc XRSPlus instrument (Bio-Rad, Hercules, CA, USA). IP was performed using antibody-coated protein A/G beads as described previously [34 (link)].