Bca quantitative kit

Forty-eight hours after transfection of cells from different treatment groups, the cells were washed three times with cold PBS (Thermo fisher, USA), and lysed on ice using whole protein lysate for 10 min. BCA quantitative kit (Thermo fisher, USA) was used for protein quantification, then 10 μl loading buffer was added and proteins were boiled at 95°C for 10 min. the proteins were loaded onto SDS-PAGE at 100 V and transferred to the NC membrane blocked with 5% BSA/TBST for 60 min. The membrane was incubated with primary antibodies at 4°C overnight and then washed with 1 × TBST solution (Solarbio, Beijing, China) at room temperature for 5 min × 3 times. the membrane was probed with HRP labeled goat-anti-rabbit IgG at room temperature for 120 min, and washed by TBST for three times. After each 20 min, the ECL kit (Solarbio, Beijing, China) was used for detecting luminescence reaction, and the protein blot was photographed and observed. The antibodies used in experiment were listed in Supplement Table 2.

Proteins in Cells or mice organs were extracted following standard procedures using the protein extraction reagents of Ripa (Millipore, Burlington, MA, USA), PMSF (CST), a protease inhibitor (Roche), and phosphatase inhibitor (Roche, Basel, Switzerland). The protein concentration was tested by the BCA Quantitative Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of total protein (30 µg) were separated by 8% SDS-PAGE gels and transferred to PVDF membranes. After being blocked in 5% skim milk for one hour, the membranes were subsequently incubated with primary antibodies at 4 °C overnight and then the secondary antibodies at room temperature for one hour. The membranes were then exposed to chemiluminescence developing agents. The antibodies used in this process were as followed: mouse anti-Collagen III (NBP1-05119, Novus), rabbit anti-Collagen I(NB600-408; Novus, Zhejiang, China), rabbit anti- β-catenin (AF6266; Affinity), rabbit anti-TFEB (abs131998; Absin, Shanghai, China), and rabbit anti-GAPDH (sc-166545; Santa Cruz Biotechnology, Dallas, TX, USA). GAPDH was used as an internal control.

48 h after cells were transfected in various groups, they were rinsed with cold PBS (Thermo Fisher, USA) in triplicate and lysed on ice for 10 min with whole protein lysate. Protein quantification was done with BCA quantitative kit (Thermo Fisher, USA). 10 μL loading buffer was supplemented, and proteins were boiled for 10 min at 95°C. SDS-PAGE was performed at 100 V. After the electrophoresis, proteins were transferred to the NC membrane at 100 mA and 120 min, sealed with 5% BSA/TBST for 60 min, followed by incubation with primary antibodies at 4°C overnight. After incubation, the membrane was washed in a shaking table with 1 × TBST solution (Solarbio, Beijing, China) 5 min × 3 times at room temperature. Goat anti-rabbit IgG labelled with horseradish peroxidase was used for hybridization for 120 min at room temperature. Membrane was washed with TBST for 20 min × 3 times and. Then, luminescence reaction was performed with ECL kit (Solarbio, Beijing, China). Protein imprinting was observed by taking photos. The assay was done in three replicates. Antibody information was exhibited in Table S3.