Lc3b antibody

L-Arginine monohydrochloride was purchased from Nacalai Tesque (Kyoto, Japan). LC3B
antibody and SEA0400 were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Myeloperoxidase (MPO) antibody was purchased from Abcam (Cambridge, UK). p62 antibody was
purchased from Cell Signaling Technology (Danvers, MA, USA).

Western Blotting was performed using standard methods. After treatment with the indicated drugs, cells were washed with cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific, 87787) supplemented with protease inhibitors cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78420). Lysates were centrifuged at 12,000 ×g for 15 min at 4 °C, and protein concentrations were determined by BCA assay (Beyotime, P0010). Proteins (30 μg) were electrophoresed on 10–12% SDS-PAGE and transferred onto a polyvinylidene difluoride membranes (Merck Millipore, ISEQ00010). After blocking with 5% skim milk in Tris-buffered saline for 2 h, the membrane was incubated with the primary antibodies for overnight at 4 °C. The following primary antibodies were used: LC3B antibody (1:1000; Sigma-Aldrich, L7543), BECN1 antibody (1:1000; MBL, PD017), ATG5 antibody (1:1000; Sigma-Aldrich, A0856), SQSTM1 antibody (1:1000; MBL, PM045), Smad2 antibody (1:800; CST, 5339S), phospho-Smad2 antibody (1:800; CST, 3108S), Smad3 antibody (1:1000; Abcam, ab28379), phospho-Smad3 antibody (1:800; Abcam, ab52903), c-Jun antibody (1:800; CST, 9165S), phospho-c-Jun antibody (1:800; CST, 3270S), GAPDH antibody (1:1000; CST, 97166S). Secondary antibodies were labeled with HRP and the signals were detected using ECL Kit (Biorad, 170-5061).