Loading buffer

Cells were lysed by SDS lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with PMSF (Solarbio, Beijing, China) and Phosphatase Inhibitor Cocktail (Beyotime Biotechnology, Shanghai, China). Proteins were diluted in loading buffer (Solarbio, Beijing, China) and isolated by SDS-PAGE, then transferred onto PVDF membranes (Millipore, Darmstadt, Germany). The membranes were incubated with the primary antibody overnight at 4 °C and then incubated with secondary antibodies specific to the primary antibody. The protein band was detected by an ECL reagent (Cytiva, USA). Images were taken using a Bio-Rad Multifunctional chemiluminescence imaging system.

The cells were lysed using RIPA solution (Solarbio, Beijing, China), which contains inhibitors of phosphatase and protease. The proteins were boiled for 10 min with loading buffer (Solarbio, Beijing, China) in preparation for western blotting. Following 7.5% SDS/PAGE resolution, the samples were blotted onto PVDF membranes (Merck, Darmstadt, Germany). After using 5% skim milk to prevent non-specific binding, monoclonal mouse anti-FLAG (1:3000, F1804; Sigma, Shanghai, China) and monoclonal rabbit anti-β-actin (1:10000, AF7018; Affinity Biosciences, San Francisco, California, USA) primary antibodies were added, and the mixture was incubated for a whole night at 4 °C. Then, the samples were treated with the corresponding horseradish peroxidase-conjugated IgG for 60 min. Lastly, the immunoreactive proteins were identified using enhanced chemiluminescence.

The RIPA lysate (Sigma, America) and PMSF (Sigma, America) were used to lyse and extract proteins at a ratio of 100:1. The concentration of the centrifuged supernatant was measured using a BCA kit (Beyotime, China), and then the protein fluid was mixed with loading buffer (Solarbio, Beijing, China) and denatured at 100 °C for 5 min. The extracted lysates were separated by 10% SDS-PAGE and transferred onto PVDF membrane. Next, the membranes were incubated with primary antibody (β-actin from Abcam, America; ARK5 form Cell Signaling Technology and Santa Cruz Biotechnology, America) overnight and then were incubated with secondary antibody (anti-rabbit IgG/anti-mouse IgG, ZSGB-Bio, China) for 1.5 h. Finally, protein expression can be observed by chemiluminescence and analyzed using Image lab.

Total protein was extracted with RIPA buffer (high) with PMSF and protein phosphatase inhibitor (all-in-one, 100×) (Solarbio, China). Proteins extracted were boiled for 10 min with loading buffer (Solarbio, China) and subjected to 10% SDS-PAGE under reducing conditions. The separated proteins were then transferred to PVDF membranes for 45 min (β-actin, pIκB, and IκB) or 90 min (TLR4, pNF-κB, and NF-κB) at 200 mA in a transfer apparatus (Bio-Rad, Philadelphia, USA). Membranes were blocked with 5% Bovine Serum Albumin (BSA) for 2 h at room temperature and incubated overnight at 4°C with diluted primary antibodies against TLR4 (1:1,000, Bioss, Beijing, China), IκB (1:1,000, CST), pIκB (1:1,000, CST), NF-κB (1:1,000, CST), and pNF-κB (1:500, Bioss) separately. The samples were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit/mouse IgG (1:1,000, Bioss) for 40 min at room temperature. To verify equal loading of the samples, the membrane was incubated with a monoclonal β-actin antibody (1:1,000, Bioss,), followed by an HRP-conjugated goat anti-mouse IgG (1:1,000, Bioss) secondary antibody. The signal was detected with Amersham Imager 600 (General Electric Company, USA) and quantified by densitometry using ImageJ software. Subsequently, the densitometry of TLR4, pIκB, and pNF-κB was normalized to β-actin, IκB, and NF-κB, respectively.

Proteins were extracted from HGFs using ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Solarbio). After being quantified by BCA (Thermo Fisher Scientific), the protein samples were mixed with loading buffer (Solarbio), separated by electrophoresis on SDS-PAGE. The proteins in the gel were transferred on a polyvinylidene fluoride (PVDF) membrane (Beyotime). The membranes were blocked with 5% skimmed milk (Solarbio) and incubated overnight at 4°C with primary antibody. The membranes were washed with Tris-buffered saline and incubated with secondary antibody for 90 min at room temperature. The PVDF membranes were subjected to chemiluminescence detection using an ECL Western Blotting Detection Kit (Solarbio).

HepG2 cells were lysed using Cellytic MT (Sigma-Aldrich, China) and the supernatant was collected after centrifugation at 12,000 rpm. The supernatant was boiled in loading buffer (Solarbio, China) at 95 °C for 5 min. Samples were examined in SDS-PAGE. After blotting on PVDF membranes (Millipore, China), the non-specific antigen was blocked with 5% non-fat milk powder and then primary antibody anti-UCP2 (89326S, Cell Signaling Technology, America) was added overnight at 4 °C. The protein was detected using a fluorescent secondary antibody and visualized using Odyssey® system (LI-COR, USA).