Anti β actin 4970

Proteins were isolated from cells and tissues using the following methods. Cells from cell culture were washed with phosphate-buffered saline and collected in lysis buffer and processed as previously outlined^{87 (link)}. Protein content was determined using the BioRad protein assay. Equal amounts of whole protein lysate were separated on SDS-polyacrylamide gels and transferred to activated polyvinylidene fluoride membranes (Millipore) Western blotting was done in keeping with standard protocols. Primary antibodies used were: anti-HELLS (ABD41, Millipore), anti-Cyclin D2 (sc-593, Santa Cruz), anti-Cyclin D1 (NBP2-32840, Novus Biologicals), anti-β-actin (4970S, Cell Signaling Technology), anti-β-tubulin (T4026, Sigma), and anti-cleaved caspase-3 (9661L, Cell Signaling Technology). Secondary antibodies conjugated to horseradish-peroxidase were: anti-mouse (715-035-150, Jackson Immuno Research), and anti-rabbit (31460, Pierce, Life Technologies). Blots were developed using Pierce ECL reagents, and chemiluminescence was detected by exposing membranes to GE-Amersham film. Blots were scanned into digital files and exposures were selected for densitometry. Densitometric analysis was done using ImageJ.

Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche). Total cell extracts were run on a polyacrylamide gel and transferred to the nitrocellulose membrane (Whatman). The following primary antibodies were used: anti-AhR antibody (BML-SA210, Enzo, 1:1,000) and anti-β-actin (4970s, Cell Signaling Technology, 13E5, 1:5,000). The immunoblot was developed using horseradish peroxidase-conjugate secondary antibodies (Bio-Rad) and the ECL Western Blotting Detection kit, as instructed by the manufacturer (GE Healthcare). Full scans in Supplementary Fig. 3.

Proteins from transfected cells were extracted with RIPA lysis buffer (Beyotime, China) supplemented with PMSF (Beyotime, China). The proteins were separated by 10–15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Then the proteins were transferred to PVDF membranes and incubated with specific antibodies. Anti-EZH2(5246S), anti-LC3B (3868S), and anti-β-actin (4970S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-HMGA1 (ab252930) and anti-DIRAS3 (ab107051) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-ULK1 (20986-1-AP) and anti-α-tubulin (11224-1-AP) antibodies were purchased from ProteinTech. Band densities were quantified by Image J software (NIH, MD, USA).