Enhanced chemiluminescence reagent

The expression of CRABP1, CRABP2 and RIG-I at protein level was detected by western blotting. As described above, the lung homogenates collected at 3, 5, 7 and 9 days after H9N2 virus infection were lysed with RIPA lysis buffer (Cell Signaling Technology, Danvers, USA), quantity of total proteins were measured by BCA kit. The lysates were centrifuged at 14,000g, and the supernatants were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Then the membranes were blocked with 3% bovine albumin and probed with monoclonals antibody to β-actin (Wuhan Sanying, Wuhan, China), CRABP1 (Wuhan Sanying, Wuhan, China), CRABP2 (Wuhan Sanying, Wuhan, China) and RIG-I (Abcam, Shanghai, China), followed by incubation with peroxidase conjugated secondary antibody (ORIGENE, Rockville, MD, USA). Proteins were visualized using enhanced chemiluminescence reagent (Cell Signaling Technology, Danvers, USA). The relative protein levels of CRABP1, CRABP2 and RIG-I to β-actin were performed by Image J software.

Frozen tissue was homogenized in lysis buffer containing protease inhibitors. The supernatant was collected after centrifugation (4°C, 12,000×g, 30 min). The cell lysates or tissue homogenates were centrifuged for 10 min (12000 g, 4°C). The supernatant was collected, and the protein concentration was calculated with a Pierce BCA protein assay kit (Pierce, Rockford, IL). Equivalent quantities of proteins were separated on 10% SDS–PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were immunostained with primary antibodies at 4°C overnight, and washed three times in TBST followed by incubation with the secondary antibody for 3 h. Band signals were detected with an enhanced chemiluminescence reagent (Cell Signaling Technology Inc., USA). The following primary antibodies were used: NR4A1 (R&D systems, USA, 1:1000) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:2000). The protein levels were normalized to those of GAPDH. The NR4A1 protein levels were grouped into high NR4A1 protein expression group and low NR4A1 protein expression group based on the median expression level.

C2C12 cells and INS-1 cells were incubated with α-Spinasterol in 6-well plates overnight. After termination of treatment, cellular proteins were extracted in RIPA buffer (Cell Signaling, Danvers, MA, USA) on ice for 20 min. Equal amounts of protein were resolved by their molecular size using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes [37 (link)]. The nitrocellulose membranes were probed with the relevant primary antibodies against P-IRS-1 (# 2385S, 1:1000, Cell Signaling), IRS-1 (# 2382S, 1:1000, Cell Signaling), P-AMPK (# 2531S, 1:1000, Cell Signaling), AMPK (# 5832S, 1:1000, Cell Signaling), GLUT-4 (# 2213S, 1:1000, Cell Signaling), P-IRS-2 (# 07-1517, 1:1000, Sigma-Aldrich), IRS-2 (# 3089S, 1:1000, Cell Signaling), PPARγ (# 2435S, 1:1000, Cell Signaling), PDX-1 (# 5679S, 1:1000, Cell Signaling), followed by horseradish peroxidase-(HRP)-conjugated anti-rabbit secondary antibodies (Cell Signaling) for 1 h on ice, and signals were detected using enhanced chemiluminescence reagent (GE Healthcare UK Limited, Buckinghamshire, UK) for 5 min at room temperature. The results were detected using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany).