Chemidoctmxrs systems

Cell lysates and fractions containing proteins were subjected to 12.5% SDS-PAGE analysis and immunoblotting. Rabbit polyclonal antibody to GFP (1:1000, MBL) and rabbit polyclonal antibody to RFP (1:1000, MBL), were used as primary antibodies, whereas horseradish peroxidase–conjugated antibody to rabbit (1:1000, Cell Signaling) was used as secondary antibodies. Immobilon Western (Millipore) was used for detection. Images were captured with ChemidocTM XRS+ systems (BIO-RAD).

Cell pellets were lysed with 1× RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and a phosphatase inhibitor cocktail (Nacalai Tesque). Nuclear proteins were collected using NE-PER Nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteins were subsequently separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were immunoblotted with the following antibodies: Rabbit anti-NF-κB p65 (phospho S536) antibody (ab28856; 1:500 dilution; Abcam), rabbit anti-NF-κB p65 antibody (#8242; 1:1000 dilution; cell signaling), rabbit anti-RelB antibody (#4922; 1:1000 dilution; cell signaling), rabbit anti-SNAIL antibody (ab180714; 1:1000 dilution; Abcam), rabbit anti-E-cadherin antibody (sc-7870; 1:500 dilution; Santa Cruz), mouse anti-Vimentin antibody (MA5-11883; 1:200 dilution; Invitrogen), mouse anti-GAPDH antibody (ab9484; 1:1000 dilution; Abcam), and rabbit anti-HDAC1 antibody (ab109411 1:1000 dilution; Abcam). The bands were visualized using Molecular Imager Gel DocTMXR+ and ChemiDocTMXRS+ Systems with Image Lab 2.0 software (Bio-Rad). Uncropped scans of the blotted gels with the weight markers are shown in Supplementary Fig. 16.

Western blot was performed as previously reported [40 (link)]. Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK). After washing in tris-buffered saline (TBS)-T, the blots were incubated with the appropriate peroxidase-coupled secondary antibody (1:2000; Anti-rabbit HRP or anti-mouse HRP, GE Healthcare Life Science). Specific proteins were detected using ECL Plus Western Blotting Reagent (GE Healthcare Life Science). The bands were visualized using Molecular Imager Gel DocTMXR+ and ChemiDocTMXRS+ Systems with Image Lab 2.0 software (Bio-Rad).