Bca kit

Tissues and cells were homogenized in RIPA lysis buffer containing protease inhibitors, and then protein concentration was quantified using a BCA kit (BestBio, Shanghai, China). Equal amounts of denatured protein were separated on a 10 or 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) for electrophoresis and then were transferred to polyvinyl difluoride (PVDF) membranes. Protein-loaded PVDF membranes were immersed into 5% skim milk for 1 h, and subsequently, membranes were incubated with primary antibodies and secondary antibodies as per the manufacturer's instructions. Antibodies were diluted using 1× Tris Buffered Saline with 0.1% Tween 20 (TBST) before incubation with membranes (all primary antibodies were 1:2,000 and secondary antibodies were 1:3,000 in this study). Subsequently, the membranes were washed three times for 10 min each with TSBT. Protein expression was detected using an Electro-Chemi-Luminescence (ECL) reagent (Tanon, Shanghai, China).

The animal total protein extraction kit (Bestbio, China) and RIPA lysis buffer (Solarbio, China) were used for protein extraction of PSCs and LX-2 cells, respectively. After quantifying protein concentration using BCA kit (Bestbio, China), 20 μg of protein samples were separated in 12% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck, Germany). The membrane was sealed with 5% skimmed milk at room temperature for 2 h, and then the primary antibodies, CE positive/negative sheep sera, polyclonal antibodies (anti-rEgE2D2 and rEgE2N), pre-immunized rat sera (1:200 v/v dilution), α-SMA, COL1A1, and GAPDH (1:5000 v/v dilution, ABclonal, China) were incubated overnight at 4 °C. After fully washing the membrane, corresponding horseradish peroxidase (HRP)-labeled secondary antibodies (goat anti-rat IgG, goat anti-rabbit IgG, and rabbit anti-sheep IgG (1:2000 v/v dilution, ABclonal, China) were added and incubated at room temperature for 1 h. Metal Enhanced DAB Substrate Kit (20 ×) (Solarbio, China) was used to visualize the bands.

Cells or tissues were lysed and proteins were separated from them, and then the concentrations were determined according to the BCA kit (Bestbio, China). After adding 20–30 μg of the above protein, the membrane was separated by electrophoresis using SDS-PAGE for 2 h, and then transferred to polyvinylidene fluoride (PVDF) membrane for about 90 min. The membrane was then immersed in 5% skimmed milk for about 2 h and operated at room temperature. Finally, incubated with primary antibodies including PI3K (1:1,000), p-PI3K (1:1,000), Akt (1:1,000), p-Akt (1:1,000), Nrf2 Nucleus (1:2,000), Nrf2 Cytoplasm (1:2000), HO-1 (1:1,000), Keap1 (1:1,000), GAPDH (1:5,000) and Lamin B1 (1:2,000) at 4°C, respectively. The next day transfer to Rabbit antibody solution (1:10,000) and incubate for 2 h. GAPDH acts as an internal protein reference. Membranes were incubated in enhanced chemiluminescence (ECL) (Thermo, United States) solution. AmershamImager 600 (GE,United States) can detect the expression of individual proteins on membranes.