Normal igg

The human USP37 cDNA was subcloned into pcDNA3.1-Flag vector or PCDH vector and USP37-C350S was made using PCR-based site-directed mutagenesis method. All other constructs were generated using standard molecular cloning methods and were confirmed by DNA sequencing. Antibodies were commercially purchased: anti-USP37 (rabbit, 18465-1-AP, Proteintech), anti-Snail (rabbit, 3879, Cell Signaling), anti-Ubi (mouse, sc-8017, Santa Cruz), anti-actin (mouse, 60008-1-lg, Proteintech), anti-N-cadherin (mouse, 33-3900, Invitrogen), anti-HA (mouse, MMS-101P, Covance), anti-Flag (mouse, F3165, Sigma), anti-Flag (rabbit, F7425, Sigma), anti-Myc (mouse, 13-2500, Invitrogen), anti-GAPDH (mouse, 60004-1-Ig, Proteintech), normal IgG (rabbit, sc-2027, Santa Cruz), and GSH beads (GE). MG132 and cycloheximide (CHX) were obtained from Sigma.

Western blot was performed using the specified antibodies. MHC (#sc-32732) and Myogenin (#sc-12732) were from Santa Cruz Biotechnology; Met (#18–7366) was from Invitrogen; P-Met (#3126), P-Akt (#9271), GAPDH (#5174), P-TYR (#9411), hALK (#3633), P-hALK (#3341) and PDGFRα (#3164) were from Cell Signaling Technology; mALK (#ab16670) was from Abcam (UK); MyoD (#M3512) was from Dako; P-MAPK (# M8159), Tubulin (#T5201), Actin (#A5060) were from Sigma-Aldrich. mALK immunoprecipitation: total cell extracts (RIPA) were 5x diluted in IP buffer (50 mM TrisHCl pH 7.9; 150 mM NaCl; 1 mM EDTA; 5 mM MgCl2; 0.1% NP-40; 20% glycerol) with 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail. Samples were precleared with equilibrated Dynabeads protein G (Invitrogen); mALK antibody and normal-IgG (Santa Cruz Biotechnology) were incubated overnight at 4°C. Mouse Phospho-Receptor Tyrosine Kinase Array (R&D Systems, Minneapolis, MN) was performed following the manufacturer’s instructions. The mean pixel density was measured using Quantity One software and expressed as a percentage of the mean pixel density of positive controls.

Cells were lysed in IP lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 7.5) and incubated with gp130/STAT3 antibody and Protein A/G Plus- Agarose (Santa Cruz Biotechnology) according to the manufacturer's instructions. Normal IgG (Santa Cruz Biotechnology) was used as a negative control. Finally, immunoprecipitates were washed four times before boiling with 4 × loading buffer.

Immunoprecipitation was performed in 3 steps; preparation of the lysates, pre-clearing of the lysates, and precipitation of the immune complexes. Firstly, MCF10A cells were lysed with RIPA buffer, and lysate samples were prepared in the same process as for Western blotting analysis. Secondly, 500 μg of cell lysates were incubated with 1 μg of normal IgG (Santa Cruz Biotech, CA) and 1 μL of protein A-agarose beads (Santa Cruz Biotech, CA) at 4°C for 30 minutes with gentle shaking and then centrifuged at 4°C and 12000 rpm for 10 minutes. The supernatant was collected, and the anti-Ku70 antibody was added and incubated overnight at 4°C with rotation. The following day, the protein A-agarose beads were added, rocked for 2 hours, and centrifuged at 4°C and 12000 rpm for 10 minutes. The pellet was collected after washing twice with RIPA buffer. Immune complexes were resuspended in 20 μL 2X SDS sample buffer and boiled at 95°C for 5 minutes. Samples were separated by SDS-PAGE electrophoresis.

HT29 cells were serum-starved overnight and treated with 100 ng/mL IL-13 for 3 h. Cells was cross-linked with 1% formaldehyde, lysed and sonicated. Sheared chromatin was subjected to immunoprecipitation overnight at 4°C with anti-STAT6 or anti-histone H3, or with normal IgG (Santa Cruz). IgG was used as the negative control antibody, whereas anti-histone H3 and the chromatin extract without any antibody treatment were used as the positive controls. Chromatin-antibody complexes were isolated using Protein A/G PLUS-Agarose (Santa Cruz). The crosslinking was reversed and genomic DNA fragments were purified and analyzed by PCR or real-time PCR (qPCR) using the following primer pair for ZEB1 promoter: 5′- CCGGTCACGTTTCAGTTT-3′ (forward) and 5′-TCCTGCTTCCCACCTCCT-3′ (reverse). All of the experiments were repeated at least three times.