Lysis buffer

MIN6 cells were seeded in 48-well plate at 5 × 10⁴ cells for 24 h, followed by 0.1 mg/ml polysaccharides or 30 mM KCl for 24 h. Then cells were incubated in KRB balanced buffer (115 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 20 mM NaHCO₃, and 16 mM HEPES; pH 7.4) containing 0.2% BSA for 2 h. Medium was then replaced with KRB containing 5.5 mM glucose, 5.5 mM glucose plus 30 mM KCl or 5.5 mM glucose plus 1.0 mg/ml of different polysaccharides for 1 h, respectively. Supernatant was collected and insulin content was measured by ELISA (Innovation Beyond Limits, Germany). The cells were lyzed with lysis buffer (Boster, AR0102) for measurement of total protein content. The insulin secretion was defined as insulin content/protein content.

For western blotting, the 0.5 cm spinal cord tissue that obtained from the epicenter of lesion sites and human umbilical vein endothelial cells (HUVECs) were homogenated and lysed by lysis buffer (BOSTER, AR0101) with the presence of protease inhibitor (Beyotime, P1005). Bradford (Ab102535) was used to quantify the concentration of protein in lysates. Equivalent amounts of protein were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5-12.5% SDS-PAGE), and then transferred onto a polyvinylidenefluoride (PVDF) membrane (Bio-Rad, 1620177). Then, the membranes were blocked with 5% skimmed milk in TBST for 2 h at room temperature, and incubated with the following primary antibodies at 4℃ overnight: TRPM2 (1:1000, NB500-242), p-CaMKII (1:1000, sc-32289), CaMKII (1:1000, sc-5306), eNOS (1:1000, sc-376751), NOX2 (1:1000, ab129068), HIF-1α (1:1000, ZEN BIO, 340462), ANG1 (1:5000, ab183701), VEGF (1:1000, sc-7269), Cleaved(C)-caspase3 (1:1000, 9664S), β-catenin (1:1000, ab32572), ZO-1 (1:1000, AF5145), Claudin-5 (1:1000, AF5216), occluding (1:1000, DF7504), P120 (1:1000, AF4684). After washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1h at room temperature. Lastly, the signals were visualized by ChemiDicTM XRS+ ImagingSystem (Bio-Rad, USA), and quantified by Image Lab.

HMCs and kidney tissues were homogenized in lysis buffer (Boster Biotechnology Co., Wuhan, China). Proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). Primary antibodies against cyclin D1, cyclin E, CDK2, CDK4, p21 (Santa Cruz Biotechnology, Dallas, TX, USA), IL-1β (ab234437; Abcam, Cambridge, UK), and α-smooth A (ab5694; Abcam) and corresponding secondary antibodies (Boster Biotechnology Co., Wuhan, China) were used. Membranes were developed and visualized using the Quantity One analysis system (Bio-Rad, Hercules, CA, USA).

The harvested cells were washed with phosphate buffered saline and lysed with lysis buffer (Boster, Wuhan, China) to obtain total cellular protein. Protein concentration was determined by using BCA Protein Assay kit (Boster, Wuhan, China). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF; Millipore Corporation, MA, USA) membrane through a Bio-Rad II System (Bio-Rad Laboratories, Inc.). Then, the membranes were sealed with 5% skimmed milk powder at room temperature for 1 h and incubated with rabbit monoclonal antibody against PI3K, P-Akt, Akt, P-mTOR, mTOR, and β-actin (1:1000; Cell Signaling Technology, MA, USA) at 4 °C overnight and goat anti-rabbit IgG at room temperature for 1 h. β-actin was used as inner loading control. The epitope was visualized by an ECL detection reagent (Millipore Corporation, MA, USA) according to the manufacturer’s instructions. The gray value was analyzed by Image-ProPlus software (Media Cybernetics, Inc., MD, USA).