Arg10112

Total protein was extracted from cells using RIPA buffer in the presence of protease inhibitor (Sigma, USA) and phosphatase inhibitor cocktail (Sigma, USA). The protein concentration was determined with a BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk (BD Biosciences, WA, USA) and 0.1% Tween-20 in Tris-buffered saline and immunoblotted overnight with primary antibodies at 4 °C with gentle shaking. Subsequently, the membranes were stained with HRP-conjugated secondary antibody. Proteins were visualized using ECL Western Blotting Substrate or Super Signal West Femto Chemiluminescent Substrate (Pierce, Rockford, IL, USA) followed by exposure to film. Antibodies used in this study are as follows: Anti-GADPH (1:1000 dilution, ARG10112, Arigo), anti-E-cadherin (1:1000 dilution, ARG66195, Arigo), anti-Vimentin (1:1000 dilution, ARG66199, Arigo), anti-ZEB1 (1:500 dilution, ARG57524, Arigo), anti-p-JAK2 (1:500 dilution, ARG57812, Arigo), anti-JAK2 (1:500 dilution, ARG57629, Arigo), anti-p-STAT3(1:500 dilution, ARG51549, Arigo), and anti-STAT3 (1:500 dilution, ARG53604, Arigo). The secondary goat anti-rabbit or goat anti-mouse (1:5000 dilution, Abcam, USA).

Cell lysates were prepared in RIPA buffer, and equal amounts of protein were loaded into NuPage 4–12% Bis-Tris Midi gels (Invitrogen) submerged in NuPage MES SDS Running Buffer (Life Technologies). The proteins were transferred onto a nitrocellulose membrane using the SureLock™ Tandem Midi Blot Module (Life Technologies) and NuPage Transfer Buffer (Life Technologies) containing 10% methanol. The membrane was blocked with 5% powdered milk in tris-buffered saline for 1 hour at room temperature. Membranes were incubated with either a mouse monoclonal anti-alpha smooth muscle actin (Abcam ab7817, 0.341ug/mL) or a rabbit monoclonal anti-vimentin antibody (Abcam ab92547, 1:5000 dilution) with a mouse monoclonal anti-GAPDH loading control (Arigo biolaboratories ARG10112, 1:5000 dilution) overnight at 4°C. Incubation with HRP secondary antibodies (Arigo biolaboratories ARG65350, 1:5000 dilution or enQuire BioReagents QAB10303, 1:15,000 dilution) was performed for 1 hour at room temperature. Signal was produced using SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and the blots were imaged on a LI-COR Fc Imager (Odyssey).

We utilized the following antibodies to perform Western blot analysis. The primary antibodies were anti-4-HNE (ARG23717, Arigo Biolaboratories, HSZ, Taiwan), anti-xCT (26864–1-AP, Proteintech, Rosemont, IL, USA), anti-GPX4 (ARG41400, Arigo Biolaboratories, HSZ, Taiwan), anti-GAPDH (ARG10112, Arigo Biolaboratories), anti-β-actin (NB600-501, Novus Biologicals, TPE, Taiwan), anti-catalase (219010, Merck, DA, Germany), anti-SOD2 (# 06–984, Merck), anti-SOD1 (ab51254, Abcam, EN, UK), anti-ULK1 (A8529, ABclonal, Woburn, MA, USA), anti-ND-1 (A5250, ABclonal), anti-UQCRC2 (A4181, ABclonal), anti-p62 (66184–1-Ig, Proteintech), and anti-Lamp2 (ab125068, Abcam). The secondary antibodies were anti-mouse IgG (7076, Cell Signaling, Danvers, MA, USA) and anti-rabbit IgG (7074, Cell Signaling).