Radioimmunoprecipitation assay lysis buffer

Immunoblotting was performed according to common standard procedures. Briefly, HT1080 cells were agitated in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich Co.) at 4°C for 30 min. Cell lysates (35 μg) were then subjected to separation using 12% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech Inc., Buckinghamshire, UK), blocking with 5% skim milk and hybridization with primary antibodies (diluted 1:1,000). After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using an electro-chemiluminescence kit (Amersham Pharmacia Biotech Inc.) according to the manufacturer’s instructions. Protein bands were observed using a CAS-400SM Davinch-Chemi imager^TM (Davinch-K).

After protein acquisition through Radioimmunoprecipitation assay lysis buffer (Sigma) and quantification by BCA Protein Kit (Sigma), western blot was implemented as previously stated [19 (link)]. The proteins were examined by incubation of primary antibodies at 4℃ overnight, including Cyclin D1 (#55506, 1:1000), matrix metalloproteinase 2 (MMP2; #40994, 1:1000), pyruvate kinase M2 (PKM2; #4053, 1:1000), DHCR24 (#2033, 1:1000), and β-actin (#4970, 1:1000). Whereafter, anti-rabbit IgG secondary antibody (#7054, 1:3000) was incubated at 25℃ for 1 hour. All antibodies were bought from Cell Signaling Technology (Boston, MA, USA). Then the special blots on the membranes were exhibited through SignalFire™ Elite ECL Reagent (Cell Signaling Technology) and data were analyzed employing the ImageJ software.

The skin samples were isolated, snap-frozen in liquid nitrogen, and immediately homogenized in ice-cold Radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich Co.) containing a cocktail of protease inhibitors (Hoffman-La Roche Ltd., Basel, Switzerland). Denatured total cell and tissue lysates were added to the ELISA analysis. VEGF, IGF-1, and TGF-β were measured with specific ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA). In brief, polystyrene 96-well plates coated with the specific antibody were incubated with the prepared standard or sample at 4°C for 2 hours. The plates were washed and incubated at 4°C for 2 hours with primary biotinylate-labeled antibody. Subsequently, the plates were washed again and incubated with streptavidin-conjugated horseradish peroxidase at room temperature for 45 minutes. After washing, tetramethylbenzidine was added to each well and incubated at room temperature for 30 minutes. Finally, sulfuric acid was used to terminate the reaction and the absorbance at 450 nm was determined with an automated microplate reader.

Radioimmunoprecipitation assay lysis buffer (Sigma) was adopted to obtain protein samples. Forty micrograms of protein samples were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After sealing for 1 h in 5% milk, primary antibodies were incubated with the membrane, including anti-Cyclin D1 (ab40754; Abcam, Cambridge, MA, USA), anti-caspase 3 (ab13585; Abcam), anti-matrix metallopeptidase 2 (anti-MMP2; ab181286; Abcam), anti- matrix metallopeptidase 2 (anti-MMP9; ab137867; Abcam), anti-GOT2 (SAB2100950; Sigma), anti-proliferating cell nuclear antigen (anti-PCNA; ab29; Abcam), and anti-β-Actin (ab8226; Abcam). After washing using Tris buffered saline–Tween 20 (Sangon Biotech) for 3 times, secondary antibody (Abcam) was added to mark the primary antibodies. Protein bands were determined using chemiluminescence reagents (Bio-Rad).

Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich). Protein concentrations were determined using a bicinchoninic acid assay reagent kit (Beyotime, Shanghai, China). Protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated in blocking solution containing 5% (w/v) nonfat milk for 1 h at room temperature. Subsequently, the PVDF membranes were incubated with primary antibodies (anti-FTO, 1:1000, Abcam, ab124892; anti-glyceraldehyde 3-phosphate dehydrogenase, 1:1000, Abcam, ab8245) at 4 °C overnight, followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. Bound antibodies were visualized with an ECL kit (Beyotime, China). Protein band intensities were quantified using ImageJ software (NIH, USA).

RAW264.7 cells were lysed with 100 μL radio immuno-precipitation assay lysis buffer (Sigma-Aldrich). Protein concentrations were measured with bicinchoninic acid assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a poly (vinylidene fluoride) membrane. The membrane was blocked with PBS containing 5% nonfat milk for 2 h at room temperature, then incubated overnight at 4°C with an anti-SR-A antibody (1:1,000; Abcam), or an anti-β-actin antibody (1:1,000; Cell Signaling, Danvers, MA, USA). The membrane was then washed three times for 5 min with 15 mL Tris-buffered saline Tween-20 and incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:1,000 dilution; Sigma-Aldrich) for 1 h at room temperature. After washing, 1 mL chemiluminescent substrate (Thermo Fisher Scientific) was added to the membrane. The signal was detected and quantified with an enhanced chemiluminescence system (ImageQuant LAS-4000 MINI; GE, Fairfield, CT, USA). All samples were normalized to β-actin. Data were represented as mean ± standard error of the mean (SEM).