Anti zeb1

Recombinant human IGF-I was purchased from R&D System (Wiesbaden, Germany). The dual IGF-IR/IR inhibitor OSI-906 was purchased from SelleckBio (USA). Specific PI3K/Akt inhibitor LY294002 was purchased from Sigma (St. Louis, MO), and specific ERK1/2 inhibitor PD98059 was purchased from Promega (Madison, WI). Proteasome inhibitor bortezomib (PS-341) was purchased from Millenium Pharmaceuticals Inc (Cambridge, MA, USA). Anti-E-cadherin, anti-Vimentin, anti-ZEB1, anti-IGF-IR, anti-phospho-IGF-IR (Tyr1131), anti-phospho-GSK-3β, anti-GSK-3β and anti-phospho-P53 (Ser15) were purchased from Cell Signaling Technology (Beverly, MA). Anti-Snail and anti-Twist2 were purchased from Abcam (Cambridge, MA). All the other antibodies were purchased from Santa Cruz Biotechnology (USA).

ChIP assays were performed essentially as described before^{22 ,26 (link),33 –44} . In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-Sp1 (Abcam, ab13370), anti-SRF (Cell Signaling Technology, 5147), (Santa Cruz, sc-585), anti-SLUG (Cell Signaling Technology, 9585), anti-ZEB1 (Cell Signaling Technology, 3396), anti-SNAIL (Cell Signaling Technology, 3879), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-598), anti-histone H3 (Millipore, 06-755), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO₃), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.

Protein samples (50 µg) were transferred to PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C, anti-E-Cadherin (1:2,000, Cell Signaling Technology, cat.no.14472), anti-N-cadherin (1:2,000, Cell Signaling Technology, cat.no.13116s), anti-Vimentin (1:1,000, Cell Signaling Technology, cat. no.5741s), anti-Zeb-1(1:2,000, Cell Signaling Technology, cat.no.70512s), anti-AR (1:2,000, Cell Signaling Technology, cat.no.19672s), anti-SOX17 (1:2,000, Abcam), anti-Notch1(1:2000, Abcam); anti-Notch2 (1:2,000, CST); anti-Notch3 (1:1,000, Abcam); anti-Notch4 (1:1000, Santa Cruze), and anti-GAPDH (1:1,000, CST, cat. no.5174s) was used as a loading control. The intensity of the protein bands was determined using Image-Pro plus 6.0.

Cells were lysed in RIPA lysis assay buffer (Pierce) supplemented with protease and phosphatase inhibitor. The samples were separated on a 4–15% SDS-polyacrylamide gel (Biorad). After transfer to PVDF membrane, probing was carried out with primary antibodies and subsequent secondary antibodies. Primary antibodies were purchased from the following commercial sources: anti-CDH1 (1:1000; Cell Signaling Technology), anti-vimentin (1:1000; Cell Signaling Technology), anti-ZEB1 (1:1000; Cell Signaling Technology), anti-SNAIL (1:1000; Cell Signaling Technology), anti-FOXC2 (1:2000; Bethyl Laboratories) and anti-GAPDH (1:10,000; Abcam). Membranes were exposed using the ECL method (GE Healthcare) according to the manufacturer’s instructions.

Immunoblotting was performed as described previously [7] (link). Anti-JARID2 (#NB100-2214, Novus Biologicals), anti-E-cadherin (#610181, BD Transduction Lab), anti-Fibronectin (SAB4500974, Sigma), anti-Vimentin (ab8069, Abcam), anti-ZEB1 (#3396, Cell Signaling), anti-ZEB2 (#61096, Active Motif), anti-phosphorylated SMAD3 (ab51451, Abcam) and anti-GAPDH (6C5, Millipore) antibodies were used. To detect the morphological changes of the cells, A549 or HT29 cells were stained with 0.4% crystal violet (Waldeck). To allow direct fluorescence of actin cytoskeleton, the cells were stained with 0.25 µM tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma). For indirect immunofluorescence, the specimens were incubated with anti-E-cadherin antibody and treated with Alexa546-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI).

AMG232 and RG7112 were purchased from APExBIO Technology. Anti-p21 (#2947), anti-PARP (#9542), and anti-ZEB1 (#3396) were purchased from Cell Signaling, anti-p53 (sc-6243), anti-MDM2 (sc-813), and anti-Nestin (sc-23927) were purchased from Santa Cruz Biotechnology and anti-beta-actin (ab8227) was purchased from Abcam. Alexa Fluor 488 anti-rabbit secondary antibody (A11008) was purchased from Thermo Fisher Scientific. Laminin (L2020) was purchased from Sigma and Hoechest33342 (H3570) was purchased from Life Technologies.