Immune blot polyvinylidene difluoride membrane

Western blot was conducted as before [27 (link)]. In brief, cells were washed with pre-cold PBS and lysed in lysis buffer (Cell signaling technology, USA) supplemented with 1 mM PMSF (Sigma-Aldrich). Samples were measured the concentration using Pierce BCA Protein Assay kit (Thermo Fisher Scientific) followed by centrifugation at 14,000 × g for 10 min at 4 °C. The protein lysate was denatured with loading buffer for 5 min at 95 °C and loaded onto 10% bis-tris protein gels. Proteins were transferred onto immune-blot polyvinylidene difluoride membrane (BIO-RAD, USA) and blocked with 5% w/v milk. The membranes were incubated with second HRP-conjugated secondary antibodies for 2 h followed by primary antibodies overnight. The protein signals were visualized using SuperSignal West Pico Chemiluminescent Substrate (BIO-RAD) and quantified grayscale measurements in Gel-Pro analyzer 4.0 (USA). The antibodies are as follows: TMEM126B (Abmart, China, 1:1000), β-actin (Santa cruz, USA, 1:2000), Anti-mouse IgG, HRP (Cell Signaling technology, 1:2000) and Anti-rabbit IgG, HRP (Cell Signaling technology, 1:2000).

Purified human α₂-macroglobulin protein (725 kDa) was obtained from Enzo Life Science (New York, USA), anti-human monoclonal mouse α₂-macroglobulin (immunoglobulin (Ig)G1 clone) from R&D Systems (Minneapolis, MN, USA), and goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) conjugate from Bio-Rad Laboratories (Hercules, CA, USA). Native PAGE™ Sample Buffer, Native PAGE Running Buffer, Dark Blue Cathode Buffer, Native Mark^™ Unstained Protein Standard, and Light Blue Cathode Buffer were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DTT, Blocking One and Peroxidase Stain 3,3′-Diaminobenzidine kit (Brown Stain) were purchased from Nacalai Tesque (Kyoto, Japan). Immune-Blot^® polyvinylidene difluoride membrane, Sequi-Blot™ membrane, Precision Plus Protein™ Dual Color Standards and Mini-PROTEAN^® TGX™ were obtained from Bio-Rad Laboratories. Perfect NT Gel, Perfect NT Gel System and SDS-PAGE Running Buffer were obtained from DRC (Tokyo, Japan), and ECL Prime Western Blotting Detection Reagent from GE Healthcare (Buckinghamshire, UK).

As described earlier^{50 (link)}, RIPA buffer was employed to lyse cells to extract total protein. After centrifugation at 14,000 rpm and 4 °C for 20 min, a BCA Protein Quantification kit was used to measure the protein concentration based on the manufacturer’s instructions. An equal volume of 2X Laemmli sample buffer was added to the cell lysates. Next, the lysates (20 μg) were exposed to SDS-PAGE, followed by boiling for 5 min and transferring them to a 0.2-μm immune-Blot polyvinylidene difluoride membrane (Cat No: 162–017,777; Bio-Rad Laboratories, CA, USA). Then, the membranes were blocked with 5% BSA (Cat No: A-7888; Sigma Aldrich, MO, USA) for 1 h in 0.1% Tween 20. Subsequently, the membranes were incubated with Anti-GRP78 (Cat No: ab21685, Abcam), anti-chop (Cat No: ab194533, Abcam), and anti-beta actin-loading control antibodies (Cat No: ab8227; Abcam) at room temperature for 1 h. After washing the membranes with TBST, incubation was performed with a secondary antibody, goat anti-rabbit IgG H&L (HRP; Cat No: ab6721; Abcam). Next, incubation with enhanced chemiluminescence was conducted for the membranes for 1–2 min. The densitometry of protein bands was calculated using Gel Analyzer version 2010a (NIH, USA). Thirty-three samples in the Western blot, 17 in the treatment group, and 16 in the placebo group were used for analysis.