Sds page electrophoresis

CS was purchased from Jimin Pharmaceutical Co., Ltd., (Jiangxi, China), TWP was puchased from Fudan Fuhua Pharmaceutical Co., Ltd., (Shanghai, China), streptozotocin was obtained from Sigma (St. Louis, MO, USA), rat urine albumin EIA assay kit was obtained from R&D Systems (Minneapolis, MN, USA), rabbit anti-rat nephrin antibodies, rabbit anti-rat podocin antibodies and goat anti-rabbit antibodies were purchased from Boster (Wuhan, China), Accu-chek Blood glucose meter was obtained from Roche Diagnostics GmbH (Mannheim, Germany). The 7150 automatic biochemical analyzer was purchased from Hitachi (Tokyo, Japan), the JEOL-1230 transmission electron microscope was obtained from JEOL (Tokyo, Japan), Vanox multifunctional microscope was purchased from Olympus (Tokyo, Japan) and SDS-PAGE electrophoresis was obtained from Bio-Rad (Hercules, CA, USA).

Total proteins were extracted from cells or tissues with RIPA lysis buffer (Sigma-Aldrich) on ice. Protein concentration were separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis (BioRad, Hercules, CA, USA) and transferred onto nitrocellulose membranes (Millipore). Blocking was performed in 5% non-fat milk for 1 h at 37 °C. After that, membranes were incubated with anti-rabbit primary antibody against PDCD4 (Sigma-Aldrich) at a 1: 1000 dilution overnight at 4 °C. β-actin served as an endogenous control and detected using a rabbit polyclonal at 1:4000 dilution (Abcam, Cambridge, MA, USA). The following day, The secondary antibody of goat anti-rabbit IgG at a 1: 1000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incubated for 1 h at room temperature. After washing, signals were visualized using LI-COR Odyssey Imaging System (LI-COR, Lincoln, NE, USA).

These experiments were performed according to previously published methods using β-actin as an internal control (Cao et al., 2013 (link); Song et al., 2018 (link)). Briefly, Caco-2 cells were inoculated into six-well culture plates. When the cells became confluent, they were treated with hypoxia or drugs and then cultured for an additional 24 h. The culture medium was discarded, and the cells were washed with cold PBS once. Then, cell lysis buffer was added, and the cells were collected and disrupted using an ultrasonic homogenizer. The cells were centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was collected and boiled in water for 5 min. An equal amount of total proteins was subject to SDS-PAGE electrophoresis (Bio-Rad, USA) and transferred onto a PVDF membrane. The PVDF membrane was blocked in 5% skim milk for 1 h and incubated with corresponding primary antibodies (ZO-1, occludin, cofilin, p-cofilin, and the internal control β-actin) at 4°C overnight. After washing four times with TBST for 15 min, the membrane was incubated with corresponding secondary antibodies at room temperature for 1 h. The membrane was washed again four times with TBS, the chemiluminescent solution was added, and the chemiluminescent signals were collected using the ChemiDoc XRS system. The results were analyzed using Quantity One software.

Liver tissue (1 mg) was placed in a microcentrifuge tube containing RIPA Lysis buffer (Applygen, Beijing, China) and homogenized on ice. After centrifugation, the supernatant was transferred to a new tube, and protein concentration was determined using the BCA Protein Assay (Applygen, Beijing, China). After lysing, 18 μg of protein was loaded onto and resolved by SDS-PAGE electrophoresis (Bio-Rad, USA) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked with 5% nonfat milk in PBS-Tween 20 (PBS-T) for 2 hours at room temperature, and the membranes were incubated overnight with the specific primary antibodies against TGF-β1 (rabbit anti-rat TGF-β1 monoclonal antibody, 1 : 1,000, AbCam, UK), Smad7 (rabbit anti-rat, Smad7 monoclonal antibody 1 : 500, Sigma, USA), or GAPDH (rabbit anti-rat GAPDH monoclonal antibody 1 : 2,000, Beyotime, China), respectively. On the next day, the membranes were washed with PBS-T three times and then incubated with sheep anti-rabbit secondary antibody conjugated with HRP (Antgene, Wuhan, China) for 1 hour at room temperature. The specific protein bands were detected with chemiluminescence in a darkroom using BeyoECL Plus (ECL Chemiluminescence Kit, ASPEN, WuHan, China) and documented and analyzed using Image Acquisition and Analysis Software (Flour Chem FC3 software, Alpha Innotech, USA).

A standard western blot analysis was used for detecting the mRNA expressions of TLR4, MyD88, NF-κB, and COX-2. Protein expressions were measured using a DCTM protein assay kit (Bio-Rad, USA). Briefly, gastric antrum tissues (150 mg) were lysed for total protein extraction; protein samples (30 μg) were subjected to SDS-PAGE electrophoresis (Bio-Rad, USA) and then transferred to a nitrocellulose membrane and blocked with TBST for 1 h; primary antibodies including TLR4 (1:800), MyD88 (1:500), NF-κB (1:1000), COX-2 (1:800), and β-actin (1:1000) were used for overnight incubation at 4°C. Subsequently, the membranes were washed thrice with TBST, followed by incubation with second antibodies for 1 h. Enhanced chemical fluorescein imaging and photographing were performed. Image analysis was performed using Graphic analysis software (Ascent software for Multiskan, Thermo ScienTific, USA), and the ratio of the gray values of the target bands to the internal reference (β-actin) was calculated.