Imager 600

Protein was extracted from AC16 cells by 100 μL containing 1 mM phenylmethyl sulfonyl fluoride (PMSF) and then isolated by 10% polyacrylamide gel electrophoresis (PAGE). And transfer it to a polyvinylidene fluoride (PVDF) film (Sigma-Aldrich Chemical Company, USA). Next, 5% skim milk was stirred and incubated at room temperature for 2 h, then the film was mixed with primary antibody (Bcl-2,ab194583; Bax,ab182733; JAK2,ab32101; STAT3,ab109085) or α-Tublin primary antibody (1:2000; 2125S, CST), etc. were treated overnight at 4 °C and then associated with secondary antibodies (1:2000; Santa Cruz Biotechnology, Dallas) incubated at room temperature for 2 h. Protein Western blot bands were detected using an electrochemical luminescence (ECL) kit (Biosharp, Shanghai, China) and Amersham Imager 600 Software.

Equal concentrations of protein samples were resolved on a 10% SDS-PAGE gel transferred to polyvinylidene fluoride membrane for immunoblotting. The blot was blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (blocking buffer). The membrane was incubated with primary antibody diluted in the blocking buffer for 1 h at room temperature or overnight at 4 °C. The membrane was washed with 0.1% TBST three times for 15 min each. The membrane was incubated with horseradish peroxidase–conjugated secondary antibody diluted in the blocking buffer for 1 h at room temperature. The membrane was washed with 0.1% TBST three times for 15 min each and developed using an ECL kit (Millipore). The blots were imaged on an Amersham Imager 600 and band intensity was quantified using Image J software.

The whole protein profile was characterized by SDS–PAGE. Briefly, the final protein concentration was quantified to be 2 mg mL^-1 by the BCA method. Then, the samples were mixed with a loading buffer (Novex) and heated for 3 min at 100 °C. Then 20 μL of each sample was loaded into BeyoGel™ Plus PAGE Precast Gel (Hepes, 4–15%, 10-well) and proteins of different molecular weights were separated by electrophoresis at 150 V for 40 min. Finally, the gels were stained with Coomassie brilliant blue and imaged.
B16F10 tumor-specific antigens were evaluated by western blotting assay. Briefly, the proteins on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the membrane was blocked with 5% bovine serum albumin and incubated with primary antibodies against sodium potassium ATPase (Na⁺/K⁺-ATPase), Glycoprotein 100 (gp100) and Melanoma antigen recognized by T-cells 1 (MART1). The secondary anti-goat or anti-mouse IgG antibody was incubated with the corresponding primary antibodies prior to ECL imaging. The chemiluminescence of the substrate was imaged by a chemiluminescence imager (Amersham Imager 600, USA).

The reactions, which were resuspended in 1× SDS protein sample buffer, were heated at 95 °C for 5 min. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membrane (Merck) via a semidry transfer method. The membrane was incubated with blocking buffer (1% skim milk and 1× Tris buffered saline with Tween 20 [TBS-T]) at room temperature for 1 h and supplemented with anti-FLAG antibodies (Sigma, #F1804; 1:10,000 dilution) or anti-HA antibodies (abcam, #ab130275; 1:10,000 dilution), anti-tubulin antibodies (Sigma, #T6074; 1:10,000 dilution), anti-NtSGS3 antibodies (29 (link)) (1:10,000 dilution), and anti-NtRDR6 antibodies (1:5,000 dilution) at 4 °C overnight. The membrane was washed with 1× TBS-T and then incubated with blocking buffer (1% skim milk and 1× TBS-T) with anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP) antibodies (MBL, #330; 1:25,000 dilution) or anti-rabbit IgG-HRP antibodies (Jackson, #111-035-003; 1:20,000 dilution) at room temperature for 1 h. After washing with 1× TBS-T, the membrane was treated with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Images were acquired using the Imager 600 (Amersham) or FUSION FX (VILBER).

The cells treated with PBS or DS were harvested, lysed, subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% skim milk for 1.5 h at room temperature, and the immunoreactivity levels were evaluated by hybridization with the following antibodies: rabbit polyclonal anti-HIF-1α (1:1,000), anti-ITGβ1 (1:500) and anti-β-actin (1:1,000) antibodies and goat anti-rabbit IgG (1:6,000). The signal was then detected by chemiluminescence using an ECL kit and exposure in a darkroom or an Amersham Imager 600 instrument. For grey value analysis with Quantity One, the target/internal relative grey scale value indicates the corresponding protein expression level. HIF-1α and ITGβ1 expression levels in cells were compared between the experimental and control groups at several time-points.