Sds polyacrylamide gel electrophoresis

Protein was extracted as described above. Proteins (30 μg) were then resolved by SDS‐polyacrylamide gel electrophoresis (Bio‐Rad) and transferred to a nitrocellulose membrane (Bio‐Rad). Blots were blocked in PBST [PBS (Phosphate buffered saline) plus 0.05% Tween 20 (Sigma Aldrich)] containing 5% instant milk and incubated with primary antibody in PBST overnight at 4°Celsius. Proteins recognized by the antibody were detected by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using a horseradish peroxidase‐coupled secondary antibody according to the manufactures’ protocol. Housekeeping genes were used to determine relative protein expression. For antibodies used: see Table 3.

Whole-cell lysates of mammalian cells and colon tissues were prepared and analyzed for immunoblot as previously performed [14 (link)]. For IP, cells were washed twice in cold PBS and lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) plus phosphatase and protease inhibitors (Roche Applied Science, Mannheim, Germany). Whole-cell extracts were incubated with the appropriate primary antibodies overnight at 4 °C. Antibody-bound proteins were precipitated with protein A/G beads according to the manufacturer’s protocol. The beads were washed four times with lysis buffer and then eluted in 2x SDS sample loading buffer. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), transferred to PVDF membranes (Merck Millipore), and detected using appropriate primary antibodies coupled with a horseradish peroxidase-conjugated secondary antibody using chemiluminescence (Thermo Fisher Scientific, MA, USA) and the LAS-4000 imager (GE Healthcare Life Sciences, Piscataway, NJ, USA).

Purified virions and recombinant proteins were mixed with an SDS sample-loading buffer, boiled for 5 min, and separated by a Tris–HCl 4%–20% (w/v) gradient SDS-polyacrylamide gel electrophoresis (BioRad). Following electrophoresis, samples were transferred to the nitrocellulose membrane (Bio-Rad). After transfer, the membrane was blocked with PBS containing 5% non-fat milk for 1 h at room temperature. Then, the purified antibodies from −20 °C stock (1:1000 diluted) were added. The next day, the membrane was washed three times with PBS containing TWEEN 20, followed by the addition of anti-rat antibodies (A9542, Sigma, 1:8000 diluted). The membrane was incubated at room temperature for 1 h and again washed three times. The immunoreactive bands were visualized using an Immun-Star AP Substrate (Bio-Rad).

Pancreatic levels of Bcl-2, Bax and pancreatic duodenal homeobox-1 (PDX-1) protein were evaluated by western blot analysis. Total proteins were extracted with RIPA lysis buffer (Beyotime institute of Biotechnology, Haimen, China) supplemented with 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Sigma, MO, USA), followed by determination of protein concentrations by bicinchoninic acid (BCA) method (Beyotime institute of Biotechnology, Haimen, China). Then protein samples were subjected to SDS-polyacrylamide gel electrophoresis (Bio-Rad, CA, USA) and transferred to nitrocellulose membrane (Millipore, MA, USA). After blocking with 5 % skimmed milk in TBS containing 0.2 % Tween-20 (TBST), the membrane was incubated overnight at 4 °C with primary antibodies against Bcl-2, Bax, PDX-1 (Santa Cruz, CA, USA) and β-actin (Beyotime institute of Biotechnology, Haimen, China), followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibody (Beyotime institute of Biotechnology, Haimen, China) for 1 h at room temperature. Bound HRP was visualized with an enhanced chemiluminescence substrate (ECL, Beyotime institute of Biotechnology, Haimen, China) and protein bands were analyzed for integrated optical density (IOD) with Quantity One software (Bio-Rad, CA, USA). The levels of PDX-1, Bcl-2 and Bax were normalized to that of β-actin.

Protein aliquots of 9 µg each were resolved by Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA). After electrophoresis, the protein samples were transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were washed three times and incubated with 5% skim milk for 1 h at room temperature, and overnight at 4 °C with the following primary antibodies: p-EGFR, p-Akt (Ser473), t-Akt, β-actin (13E5) (Cell Signaling Technology, Danvers, MA, USA), t-EGFR, p-Erk1/2 (Thr202/tyr204), t-Erk1/2 (R&D systems, Minneapolis, MN, USA). After washing three times, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated species-specific secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Immunoreactive bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany).

Dissected hippocampi were immediately immersed individually in a lysis buffer and sonicated for 10 s (n = 3 mice per group). Sodium dodecyl sulfate (SDS) sample buffer (4×) was added to each homogenized sample, and the samples were heated at 100 °C for 10 min. Immunoblotting was performed as described previously [55 (link)]. Briefly, the resolved proteins were separated with SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred onto membrane by using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was incubated with rabbit anti-Nrf2 (1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After extensive washing and incubation with secondary antibodies for 2 h at room temperature, signals were developed using a chemiluminescence kit (SuperSignal^® West Pico PLUS; Thermo Fisher Scientific) and read on a ChemiDoc MP Imaging System (Bio-Rad). To quantify Nrf2 expression, the membrane was re-probed with an antibody for β-actin (1:10,000; Sigma-Aldrich). Several exposure times were used to obtain signals within the linear range, and the bands were quantified using the ImageJ software (NIH, Bethesda, MD, USA).