Anti herg

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.

Rabbit polyclonal anti-hERG was purchased from Alomone (Alomone, Israel). Anti-CNX antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CRT antibody was purchased from Abcam (Abcam, U.S.A.). Rabbit polyclonal anti-ATF6 was purchased from Active Motif (Active Motif, U.S.A.). The proteasome inhibitor of N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) was purchased from Calbiochem, Propranolol (Prop) was purchased from Sigma (Sigma, U.S.A.).

Cell lysates were prepared in Buffer D (20 mM HEPES, 125 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM dithiothreitol, 1 mM EDTA, and 1% Triton X-100 (pH 7.6)) supplemented with 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 4 μg·mL⁻¹ aprotinin. Protein lysate (30 or 60 μg) was separated by 7% or 10% SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Veenendaal, The Netherlands). Ponceau staining was used as a loading control for subsequent quantification. Blots were blocked for 1 h with 5% Protifar dissolved in Tris-buffered saline/Tween 20 (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% (v/v) Tween-20). For protein detection, the membrane was incubated with anti-K_IR2.1 (1:1000; Sigma-Aldrich, St. Louis, MO, USA), anti-GFP (1:500; Santa Cruz Biotechnology, Heidelberg, Germany), anti-hERG (1:2500; Alomone Labs, Jerusalem, Israel), or anti-sodium channel primary antibody (1:2000; custom-made [81 (link)]). A peroxidase-conjugated Goat anti-Mouse (Jackson ImmunoResearch, West Grove, PA, USA) or Goat anti-Rabbit (Jackson ImmunoResearch, West Grove, PA, USA) antibody was applied as the second primary antibody. Final detection was performed using the Standard ECL procedure (Amersham Bioscience, Buckinghamshire, UK). Quantification was done by Image Lab software version 6.1 (Bio-Rad Laboratories, Veenendaal, The Netherlands).