Sumo2 3

Total protein was extracted from cells as previously described. Cells were lysed in RIPA buffer (Beyotime Biotechnology; China) containing 1% protease inhibitors (Roche Diagnostics, Mannheim, Germany) or 20 mM N-ethylmaleimide as appropriate (Sigma-Aldrich; St. Louis, MO, U.S.). The lysates were separated on an 8% or 12% SDS-PAGE gel and then transferred to nitrocellulose filter membranes. After being blocked in 5% non-fat milk, the membranes were incubated with the following primary antibodies: anti-PML (1:500 dilution; MBL, Nagoya, Japan), SUMO-1 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.), SUMO-2/3 (1:500 dilution; Abcam, Cambridge, U.K.), UBC9 (1:500 dilution; Cell Signaling Technology, Beverly, MA, U.S.); RNF4 (1:500 dilution; Abcam, Cambridge, U.K.); anti-TGF-β1 (1:500 dilution; Cell Signaling Technology, Beverly, MA, U.S.) and anti-HERG (1:500 dilution; Alomone labs, Jerusalem, Israel). GAPDH (1:10,000 dilution; Research Diagnostics, Concord, MA, U.S.). Goat anti-rabbit (1:10,000 dilution; Alexa Fluor 700 conjugated; Molecular Probes/Life Technologies) served as the secondary antibody. Immunoblots were imaged using an LI-CORE Imaging System (LI-COR Biosciences, Lincoln, NE, U.S.) and Odyssey software was used for quantification of bands normalized to GAPDH.

Antibodies used in this study are listed below where respective dilutions are mentioned in the parentheses:
α-actinin, mouse monoclonal, Sigma-Aldrich (1:400); SUMO2/3, rabbit monoclonal, Cell signaling (1:1000); SUMO2 + 3, mouse monoclonal, Abcam (1:1000); Calcineurin A, mouse monoclonal, BD Bioscience (1:250); GAPDH, mouse monoclonal, Sigma-Aldrich (1:20,000); Histone H3, rabbit polyclonal, Cell-signaling (1:1000); α-Tubulin, mouse monoclonal, Sigma-Aldrich (1:8000); Ubiquitin, mouse monoclonal, Millipore (1:1000); STAT1, rabbit monoclonal, Cell signaling (1:1000); p-STAT1, rabbit monoclonal, Cell signaling (1:1000); STAT3, rabbit monoclonal, Cell signaling (1:1000); p-STAT3, rabbit monoclonal, Cell signaling (1:1000); F4/80, rat monoclonal, Dianova (1:500); HA, mouse monoclonal, Sigma-Aldrich (1:20,000); HectD3, rabbit polyclonal, Mybiosource (1:1000); V5, mouse monoclonal, Biozol (1:1000).

Human 293T and HeLa cell cultures were maintained in 6-cm dishes with DMEM supplemented with 10% fetal bovine serum, then treated with 2.5 mg/mL of 1,10-phenanthroline or with DMSO in serum-free DMEM. Medium was discarded and cells were washed with ice-cold PBS. Cells were scraped and chilled lysis buffer (0.25 mL per plate; 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 8.0; 2.5 mg/mL N-ethylmaleimide) was added to the cells, which were then scraped and transferred to microfuge tubes. For the 30-min treated samples, cells became less adherent and were therefore transferred to microfuge tubes prior to washing in PBS and the addition of the lysis buffer. Samples were placed on a rotator at 4 °C for 30 min, then spun at maximum speed in a refrigerated microfuge (4 °C). Supernatants were transferred to fresh tubes and analyzed by SUMO1 and SUMO2/3 immunoblots. Antibodies were used at the following dilutions: 1:250 SUMO1 (DSHB); 1:125 SUMO2/3 (Abcam). For loading control, lysates separated by SDS-PAGE were stained with Coomassie brilliant blue.