Pre cooled ripa buffer

As previously described [22 (link)], prepared cells were added with pre-cooled RIPA buffer (Beyotime, Shanghai, China) and supplemented with protease and phosphatase inhibitors. The resulting samples were denatured by heating to 100 °C for 15 min. The BCA™ kit (Thermo Fisher Scientific, USA) was used to measure protein concentration. The same quantity of protein was increased in size and placed onto a gel made of 12 % sodium dodecyl sulfate-polyacrylamide, then moved to a PVDF membrane. The membrane was placed in a solution of tris-buffered saline with 5 % (w/v) milk in tween and incubated for 1.5 h. The primary antibody was included and left to incubate at a temperature of 4 °C for the entire night. Afterwards, the membrane was subjected to the secondary antibody for an additional hour. Enhanced chemiluminescence was utilized to detect the immunoblots ultimately. Primary antibodies were GYS1 (Proteintech, 10566-1-AP) and NCKAP1 (Proteintech, 12140-1-AP).

Western blot was used to analyze the protein expression levels of COL1, RUNX2, OPN and OSX. The hPDLCs were treated as described for the qRT-PCR assay. The cells were disrupted in pre-cooled RIPA buffer (Beyotime) after being treated with EGCG for 14 d. The protein content in the extracted cell lysates was determined using a Pierce™ BCA Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. The obtained proteins (20 μg) were separated by 8~12% SDS-polyacrylamide gels. Then, the proteins were transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, MA, USA) with a pore size of 0.45 μm, followed by blocking with 5% fat-free dry milk for 3 h. Subsequently, the membranes were incubated with individual primary antibodies at 4 °C overnight. The following antibodies were used: anti-COL1 (ab138492, Abcam, MA, USA), anti-RUNX2 (ab23981, Abcam), anti-OPN (ab91655, Abcam) and anti-OSX (ab22552, Abcam). The membranes were then incubated with the secondary antibody (ab205718, Abcam) conjugated with horseradish peroxidase for 1 h. The protein bands on the PVDF membranes were then stained using an enhanced chemiluminescence reagent. The stained bands were visualized using the Image Studio Lite software (Media Cybernetics Inc., Bethesda, MD, USA) and quantified by comparing the band intensities to that of GAPDH.

After visceral adipose samples were collected, total protein was extracted using pre-cooled RIPA buffer (Beyotime, China) at 4°C to detect the levels of IL-1β (MLB00C, R&D, United States) and IL-10 (ab108870, Abcam, United States) by an ELISA kit. After 30 min standing of mice blood, serum samples were obtained by 3,500 g centrifugation for 15 min at 4°C. The levels of IL-1β (MLB00C, R&D, United States) and IL-10 (ab108870, Abcam, United States) were measured by the ELISA kit.