Sds polyacrylamide gel

Cells were lysed in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate and protease inhibitor cocktail tablet (Roche Diagnostics, Basel, Switzerland). The protein content in each sample was determined using a Bio-Rad Protein assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of protein were separated by electrophoresis on 7.5~10% SDS-polyacrylamide gels (Beyotime Institute of Biotechnology, Haimen, China). The electrophoresed proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.). Subsequent to overnight incubation at 4°C in a blocking buffer [5% non-fat dry milk and 0.05% Tween-20 in Tris-buffered saline (TBST)], the membranes were immunoblotted with antibodies against rabbit anti-human polyclonal Cav-1 and mouse anti-human monoclonal TIGAR (cat. nos. sc-894 and sc-377065; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. Following three washes with TBST for 5 min each, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, sc-2004 and goat anti-mouse IgG, sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Finally, the membranes were developed using Beyotime ECL Plus (Beyotime Institute of Biotechnology).

In each group, proteins (20 µg for cytoplasmic protein and 5 µg for nuclear protein) were separated on 10% SDS-polyacrylamide gels (Beyotime Institute of Biotechnology) and then transferred onto polyvinylidene difluoride membranes (EMD Millipore). Subsequently, the membranes were blocked with Tris buffered saline with 5% Tween-20 containing 5% non-fat milk for 1 h at room temperature. The levels of Keap1 (cat. no. WL03285; Wanleibio), Nrf2 (cat. no. WL02135; Wanleibio) and HO-1 (cat. no. WL02400; Wanleibio) were detected by immunoblotting using specific primary and secondary antibodies. β-tubulin (cat. no. 2128S; Cell Signaling Technology, Inc.) and Lamin B (cat. no. 13435S; Cell Signaling Technology, Inc.) were used as controls to ensure equal loading of cell lysates. All primary antibodies were rabbit antibodies and were used at a concentration of 1:1,000. The secondary antibody (cat. no. IH-0011; Beijing Dingguo Changsheng Biotechnology Co., Ltd.) used was an anti-rabbit antibody and was used at a concentration of 1:5,000. The primary antibody was incubated at 4˚C for 20 h and the secondary antibody was incubated at room temperature for 1 h. ECL (cat. no. 32106; Thermo Fisher Scientific) was used to develop the bands. The band intensity was analyzed using ImageJ 1.8 software. Data were analyzed by one-way analysis of variance (ANOVA).

Rats were administered 4% chloride hydrate and sacrificed. Brain tissues were removed and
separated on ice. Peri-injury cortical tissues, which contained the target protein, were
immediately frozen at –80°C before use. The sheared tissues were then dissolved by RIPA
lysis buffer (Beyotime, Shanghai, China) and phosphatase inhibitors (Abcam, Cambridge, UK)
and centrifuged at 13,000 rpm at 4°C for 30 min. The protein concentration of the
collected supernatant was tested by a Pierce^TM BCA Protein Assay Kit
(Thermo-Fisher Scientific, Loughborough, UK). The resulting samples were separated by
electrophoresis on 10% SDS-polyacrylamide gels (Beyotime, Shanghai, China). After transfer
to PVDF membranes (Millipore, Bedford, MA, USA), 5% nonfat milk was used to block the
membranes at room temperature for 2 h. The membranes were then incubated with the
following primary antibodies overnight at 4°C: rabbit anti-BNIP3 L (1:400, Abcam), rabbit
anti-LC3B (1:1000, Abcam), rabbit anti-SQSTM1 (1:1000, Abcam), chicken anti-Albumin
(1:1000, Abcam), and rabbit anti-GAPDH (1:10000, Abcam). A corresponding horseradish
peroxidase (HRP)-conjugated secondary antibody was used to react with the appropriate
primary antibody. Protein bands were developed in an enhanced chemiluminescence system
(ECL, Pierce, Waltham, MA, USA) and then analyzed by using ImageJ software.

For protein extraction from hPSCs, a total of 15×10⁴ hPSCs were seeded into each well of a 6-well plate. After treatment with MitoQ or vehicle for 48 h, proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China). For pancreatic tissue protein, the pancreatic tissue samples were diluted 10 times using RIPA and homogenized. Next, the samples were centrifuged at 12,000 g or 15 min at 4 °C and the concentrations of various proteins in the supernatant solution were measured using the BCA protein assay kit (Beyotime, Shanghai, China). To separate the proteins, 10% SDS–polyacrylamide gels (Beyotime) were used. Next, the proteins were transferred onto polyvinylidene difluoride (PVDF, Millipore, Billerica, MA, USA) membranes, and after blocking with 5% skimmed milk, the membranes were incubated overnight with primary antibodies at 4 °C (the list of primary antibodies used is provided in Supplementary Table 3. Thereafter, the membranes were incubated with a type of near-infrared fluorescent dye-conjugated secondary antibody (LI-COR, Lincoln, NE, USA) at room temperature for 1 h, and finally, scanned using an Odyssey infrared fluorescence imaging system (LI-COR). The images obtained were further analyzed using Image J software (NIH).

Primary BMDCs were harvested and stimulated with different concentrations of EPZ015666 (Selleck) in the presence or absence of LPS (100 ng/mL) (Sigma). After the indicated times, BMDCs were collected and lysed with RIPA buffer containing protease and phosphatase inhibitors (Beyotime, Shanghai, China). The protein samples (25 mg) were separated on 10% SDS-polyacrylamide gels (Beyotime) before being transferred to PVDF membranes (Beyotime). After blocking with 5% nonfat milk in TBST for 1 h, the membranes were incubated with primary antibody against PRMT5 (Abcam) (Cell Signaling Technology, MA, USA) overnight at 4 °C. After three washes with TBST on the next day, the membranes were incubated with secondary HRP-labeled anti-rabbit/anti-mouse antibody (Beyotime). Immunoreactivity was visualized using a Fusion Pulse 6 system (Vilber Lourmat, France).