Anti slug antibody

Proteins were isolated from cells as previously described, and were then used in the Western blot analyses (10 μg/lane) [44 (link), 45 (link)]. The membrane was probed with the following antibodies: anti-FGF2 antibody (#sc-0079, 1:1000; Santa Cruz Biotechnology, Heidelberg, CA, USA), anti-TYMS antibody (ab58287, 1:1000; Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (1:1000; Abcam), anti-N-cadherin antibody (1:1000; Abcam), anti-vimentin antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-slug antibody (1:1000; Cell Signaling Technology), anti-phosphorylated (p)Smad antibody (1:1000; Abcam), anti-twist antibody (1:1000; Abcam), anti-phospho-p44/42 MAPK (Tyr202/Tyr204) antibody (1:1000; Cell Signaling Technology), anti-p44/42 MAPK antibody (1:1000; Cell Signaling Technology), anti-phospho-Akt antibody (1:1000; Cell Signaling Technology), anti-Akt antibody (1:1000; Cell Signaling Technology), anti-MEK antibody (1:1000; Cell Signaling Technology), anti-phospho-MEK antibody (1:1000; Cell Signaling Technology), anti-phospho-EGFR antibody (Tyr1068) (1:1000; Cell Signaling Technology), and anti-EGFR antibody (1:1000; Cell Signaling Technology), and anti-β-actin (1:5000; Sigma, Saint Louis, MO, USA) was used as a loading control. Each experiment was repeated independently at least three times and one representative blot was selected for the figures.

Anti-FBXO31 antibody (#86137; 1:1000) antibody was purchased from Abcam. Anti-AuroraA antibody (#14475; 1:1,000), anti-Slug antibody (#9585; 1:1000), anti-C-myc antibody (#5605; 1:1000), anti-p-p44/42 MAPK (Erk1/2) (#4370; 1:1000) and anti-tubulin antibody (2128 S; 1:2000) antibodies were all obtained from Cell Signaling Technology. Anti-SIRT2 (66410-1-Ig; 1:1500), anti-METTL3 (15073-1-AP; 1:1500), anti-METTL14 (26158-1-Ap; 1:1500), Anti-Flag tag (20543-1-AP; 1:1500), anti-HA tag (51064-2-AP; 1:1000), Anti-β-actin (20536-1-AP; 1:2000) and anti-His tag (66005-1-Ig; 1:1,000) antibodies were all bought from Proteintech. Peroxidase-conjugated anti-mouse secondary antibody (70-GAM007, 1:5000) and peroxidase-conjugated anti-rabbit secondary antibody (70-GAR0072; 1:5,000) were purchased from MultiSciences Company.

The human breast cancer cell lines MCF-7 and MDA-MB-231 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Antibodies were from various sources: anti-E-cadherin antibody (BD Biosciences, Bedford, MA), anti-SLUG antibody (Cell Signaling, Beverley, MA), anti-SNAIL1 and anti-α-tubulin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody used for immunofluorescence staining in MCF-7 and MDA-MB-231 cells was anti-E-cadherin (BD Biosciences, Bedford, MA).

Cell culture materials were obtained from Invitrogen (Burlington, Ontario, Canada). The reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI) and the PureLink™ HiPure Plasmid DNA Purification Kit were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against PARP and Twist were purchased from GeneTex (Beverly, MA, USA). Anti-HER-2, anti-phospho-Akt (Ser473), anti-E-cadherin, anti-vimentin, anti-Snail, and anti-Slug antibodies were purchased from Cell Signaling (Beverly, MA, USA). Primary antibodies against ILK, phosphor-ILK (Thr173), phospho-mTOR (Ser2448), and phospho-GSK3β (Ser9) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against YB-1 and β-Actin were purchased from Millipore (Temecula, CA, USA). The antibody against HIF-1α was purchased from BD Biosciences Clontech (San Jose, CA, USA). For Western blotting, the secondary antibodies of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Millipore (Temecula, CA, USA), and enhanced chemiluminescence (ECL) reagents were purchased from Sigma-Aldrich. Emodin, AE, and rhein were purchased from Sigma-Aldrich. The RNAi Consortium of YBX-1 was selected by the National RNAi Core Facility.

SiHa and C33A cells were subjected to ChIP using the EZ-ChIP Assay kit (Millipore). Briefly, the cells were treated with 37% formaldehyde to crosslink proteins, and the reaction was terminated with 0.125 M glycine. After sonication, chromatin–protein complexes were immunoprecipitated with 5 μg of anti-Slug antibodies (#9585, Cell Signaling Technology) or 1 μg of mouse IgG. Real-time PCR was performed to amplify the regions of interest or internal negative control regions. Each sample was assayed in triplicate, and the fold enrichment ratio was calculated as the value of the ChIP sample versus the corresponding input sample. Samples that yielded a two-fold enrichment or better were considered positive targets. The primers used for these studies are listed in Table S3.