Ecl prime solution

Cells were lysed with radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.8, 150 mM NaCl, 0.5% deoxycholate, 1% IGEPAL CA-630) and protease inhibitor cocktail (Roche). Lysates were obtained after centrifugation at 15,000 rpm for 20 min, by transferring the supernatant. Protein concentration was quantified with BCA protein assay kit. Overall protein sampling procedures were done at 4 °C. Prepared protein samples were analyzed with SDS-PAGE followed by a western blot procedure. Proteins were transferred into the PVDF membrane, and it was blocked with 2% BSA in TBST over 1 h at room temperature (r.t.). Primary antibodies were treated overnight at 4 °C [Anti-LC3B; 1:2000, anti-ATG5; 1:1000, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 1:2000, anti-DGAT1; 1:1000, anti-DGAT2; 1:1000, anti-SOAT1; 1:500, anti-SOAT2; 1:500] followed by washing with TBST. HRP-labeled anti-rabbit IgG or HRP-labeled anti-mouse IgG secondary antibody (1:5000) were treated at r.t. for 1 h. After washing with TBST, the membrane was developed by Amersham ECL prime solution. The chemiluminescent signal was measured by the ChemiDoc^MP imaging system.

Cells were lysed in RIPA buffer (ThermoFisher Scientific) on ice for 15 min before centrifuging at 15,000 rpm for 15 min at 4 °C. Extracted proteins were quantified using the Pierce BCA assay (ThermoFisher Scientific) before loading 8 ug of total protein with 10% v/v LDS sample buffer (Life Technologies, Carlsbad, CA, USA) onto a 4–12% Bis-Tris pre-cast protein gel (Sigma-Aldrich). Samples were run at 150 V on ice for approximately 1 h before transferring to nitrocellulose membranes at 40 V for 1.5 h. The membranes were incubated in 5% skim milk for 1 h before incubation overnight at 4 °C in mouse anti-human LAMA5 (CL3118, Novus Biologicals, Centennial, CO, USA), mouse anti-human Hey2 (ab167280, ABCAM) or GAPDH (D16H11, Cell Signalling Technology, Danvers, MA, USA) diluted at 1:500 in 1% skim milk. After incubation in secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), the membranes were incubated in ECL Prime solution (Amersham, Amersham Pl, Little Chalfont, Amersham, United Kingdon) and visualised using the Odyssey Fc imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein bands were quantified using ImageJ.

RAW 264.7 macrophages were extracted with RIPA lysis buffer containing phosphatase and protease inhibitor cocktail (GenDEPOT, Barker, TX, USA), and total protein concentrations were then determined using a BCA protein assay kit (Thermo Scientific). Equal amounts of total proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were then blocked in 5% skim milk for 2 h at RT and incubated overnight at 4°C with primary antibodies against p38, iNOS, COX-2, ERK1/2, JNK, Nrf2, Lamin B, HO-1, β-actin, and the phosphorylated forms of p38, NF-κB, IκB-α, ERK1/2, and JNK. Membranes were then rinsed and incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h at RT. After rinsing, bands were visualized using ECL prime solution (Amersham Bioscience, Buckinghamshire, UK). Blots were quantified by densitometry using scientific imaging software (ImageJ 1.42; NIH, Bethesda, MD, USA) after normalizing versus β-actin, which was used a loading control.