Chemidoc xrs gel imaging system

Proteins were extracted from heart homogenate with buffer (50 mM Tris-Cl, 150 mM NaCl, 100 μg/mL phenylmethylsulfonyl fluoride, protease and phosphatase inhibitor cocktail and 1% Triton X-100). After centrifugation at 12,000 g for 20 min at 4 °C, the supernatant was used for Western blot analysis. In brief, equal amounts of protein (10–40 μg) and molecular weight markers were loaded into the wells of SDS-PAGE gels. After running for 1–2 h at 100 V, the gels were transferred to PVDF membranes. Then, the membranes were blocked with blocking buffer (5% nonfat milk, 50 mmol/L Tris-HCl, 150 mmol/L NaCl and 0.1% Tween 20) for 1 h at room temperature. Next, the membranes were incubated with the appropriate dilutions (1:1000–1:2000) of primary antibody in blocking buffer overnight at 4 °C. After thorough washing, the membranes were incubated with the recommended dilution (1:5000–1:10,000) of conjugated secondary antibody in blocking buffer at room temperature for 1 h. After thorough washing and incubation with the chemiluminescent substrate, the membranes were placed into the ChemiDoc™ XRS+ Gel Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for visualization.

The human p-RTK array kit (R&D System, Inc.) was used to detect the level of RTK phosphorylation. Cells were treated with dimethyl sulfoxide (DMSO) or drugs for 36 h and then lysed with lysis buffer, 300 μg proteins were processed according to the manufacturer’s protocols. Briefly, these arrays were incubated with cell lysates overnight with shaking at 4 ℃ and then washed with washing buffer for three times. Arrays binding with target protein were then incubated with anti-p-tyrosine-HRP detection antibody with rocking for 2 h at room temperature. The arrays were washed and incubated chemiluminescent reagent, and finally exposed by ChemiDoc XRS+ gel imaging system (Bio-Rad, USA).

For Western blotting, equal amounts of protein were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The resulting blots were blocked with 5% non-fat milk (in Tris-buffered saline with Tween 20) and incubated with primary antibodies overnight at 4°C. Then, protein bands were detected by incubating with secondary antibodies for 1–2 h at room temperature and visualized with enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) by Chemi Doc XRS + gel imaging system (Bio-Rad, USA). Densitometry analysis was performed using Quantity One software, and the band intensities were normalized to those of β-actin and GAPDH.
Immunoprecipitation was performed as previously described (Zhu et al., 2013 (link)). Briefly, the cell lysates were centrifuged to remove the cell debris and then were incubated with beads (Abmart) for 1–2 h. Endogenous FLCN and HIF2α were immunoprecipitated using an anti-FLCN or anti-HIF2α polyclonal antibody. The beads were boiled after extensive washing, resolved via SDS-PAGE, and analyzed via immunoblotting. The protein concentration was detected using the Odyssey system.

To evaluate interaction between PRLR/GHR and Ab2β, non-denaturing electrophoresis was done. The extracellular domain of PRLR (PRLR-ECD) or GHR (GHR-ECD) was labeled with 5-iodo-acetamido fluorescein (IAF) (Beck et al., 2002 (link)). For binding experiments between PRLR/Ab2β or GHR/Ab2β, native gel electrophoresis was done and the gels were imaged using Biorad’s ChemiDoc XRS Gel Imaging System.

LPS (Lipopolysaccharides from Escherichia coli O555: B5, L2880-100MG) was purchased from Sigma (USA) (Batch number: 017M4112V). Dexamethasone (DXM) was purchased from Anhui Golden Sun Biochemical Pharmaceutical Co., Ltd. (Anhui, China) (batch number: 15032521). 7100 Automatic biochemical analyzer (Hitachi, Japan), Bio-Rad electrophoresis unit (USA), and Bio-Rad ChemiDocXRS + Gel Imaging System (USA) were used in this study. LEICA RM2235 paraffin slicer (Germany) was used to generate sections for microscopy. LEICA DM2500 Optical Microscope (Germany) was used to measure neutrophil invasion. Urea nitrogen (BUN) (R1 TG836, R2 TG837), total protein (TP) (TH619), albumin (ALB) (TF126), aspartate aminotransferase (AST) (R1 AR792, R2 TG862), alanine aminotransferase (ALT) (R1 AR794, R2 TH622), lactate dehydrogenase (LDH) (R1 AR796, R2AP318) and alkaline phosphatase (ALP) (R1 TF168, R2 AR800) were all purchased from Japan Pure Pharmaceutical Industry Co., Ltd. in Shanghai, China. Creatinine (Cre) (710241 H) and creatine kinase (CK) (708021 G) were purchased from Beijing Leadman Biochemical Co., Ltd. (Beijing, China).