Bca protein assay kit

After extraction of total protein by RIPA buffer containing phosphatase inhibitors (Biotech Well, Shanghai, China), the concentration of proteins was measured by The BCA protein assay kit (Beyotime, Shanghai, China). Equivalent amounts of protein in each sample were separated by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and followed by transferred onto a polyvinylidenedifluoride (PVDF) membrane (BIO-RAD, United States). Subsequently, these protein was probed with the respective primary antibodies at 4°C overnight, including USP13 (1:1,000, No. PA5-106761, Thermo Fisher Scientific, Massachusetts, United States), AMPK (1:1,000, No. 4150, Cell Signaling Technology, Beverly, MA, United States), LC3 (1:1,000, No. 4599, Cell Signaling Technology, Beverly, MA, United States), Beclin 1 (1:1,000, No. 3495, Cell Signaling Technology, Beverly, MA, United States), VPS34 (1:1,000, No. ab124905, Abcam, Cambridge, United Kingdom) and GAPDH (1:3,000, No. 5174, Cell Signaling Technology, Beverly, MA, United States) followed by addition of appropriate HRP-conjugated goat anti-rabbit IgG (1:5,000, No. S0001, Affinity Biosciences, Beijing, China) at 37°C for 1 h. GAPDH was used as an endogenous reference. The protein signal was analyzed by ECL assay kit (enhanced). Each trial was performed in triplicate.

Cells were lysed by Radio Immunoprecipitation Assay (RIPA) Lysis buffer (Beyotime, Shanghai, China), and the quantity of protein was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Extracted proteins were loaded, separated by SDS-PAGE, and blotted onto PVDF membranes (Millipore, MA, USA). Membranes were then blocked using 5% non-fat dry milk and incubated with primary antibody at 4°C overnight. The following primary antibodies were used: anti-E-cadherin (1:2000; #60335-1-Ig, Proteintech, Wuhan, China), anti-CALB2 (1:2000; #66496-1-Ig, Proteintech, Wuhan, China), anti-MMP9 (1:2000; #10375-2-AP, Proteintech, Wuhan, China), and anti-GAPDH (1:5000; #5174, Cell Signaling Technology, MA, USA). The membranes were then exposed to the secondary antibody. Finally, using the Omni-ECL chemiluminescent reagent (#SQ201, epizyme, Shanghai, China), protein blots were identified with the Tanon 5200 Multi Chemiluminescent Imaging System (Tanon, Shanghai, China).

RIPA Lysis Buffer (Beyotime) was used to extract total protein, and BCA Protein Assay Kit (Beyotime) was used to quantify the protein. Afterwards, protein samples (30 µg) were separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Beyotime). Next, the membranes were blocked with skimmed milk for 2 h. After incubating with primary antibodies against Bcl-2 (26Kda, 1:2,000, BA0412, Boster, Wuhan, China), Bax (20Kda, 1:1,500, BA0315-2, Boster), Cleaved-caspase 3 (17Kda, 1:1,000, AC033, Beyotime), FOXO1 (82Kda, 1:2,000, AF603, Beyotime), or GAPDH (36Kda, 1:2,000, A00227, Boster), the membrane was then incubated with HRP Conjugated AffiniPure Goat Anti-rabbit/mouse IgG (H + L) (1:10,000, BA1056, Boster). The protein signals were visualized using BeyoECL Star (Beyotime). EasySee Western Marker (25-90Kda, DM201-01, Transgen Biotech, Beijing, China) was used as a molecular weight standard.

Cells were lysed on ice by modified RIPA buffer (P0013B; Beyotime, Shanghai, China) to obtain total protein extract that was quantitated by BCA protein Assay kit (P0010; Beyotime, Shanghai, China), and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes that were then blocked in 5% milk diluted with TBST for 1 h at room temperature, probed with primary antibodies, such as BMAL1 (Abcam, 1:1000), CLOCK (Abcam, 1:1000), PER2 (Abcam, 1:1000), CRY2 (Abcam, 1:1000), GAPDH (Santa Cruz Biotechnology Inc., 1:10000), FVII (Santa Cruz Biotechnology Inc., 1:1000), and FXII (Santa Cruz Biotechnology Inc., 1:500), at 4°C overnight. Then, the membranes were incubated with a secondary goat anti-rabbit antibody (1:2000) on the following day for 1 h at room temperature. ECL enhanced chemiluminescence substrate kit (Millipore) was used for imaging and quantitation of the immunoreactive bands by the Image J software.

Proteins were extracted from cells using Radio‐Immunoprecipitation Assay buffer (RIPA, Beyotime Institute of Biotechnology), and the concentration of proteins was calculated by a BCA protein assay kit (Beyotime Institute of Biotechnology). An equal amount of proteins (20 μg/lane) was subjected to 12% SDS‐PAGE, followed by an electrophoretic transfer onto a polyvinylidene fluoride membrane. After being blocked with 5% non‐fat milk dissolved in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 hour at room temperature, membranes were incubated with rabbit polyclonal antibodies against EZH2 (ab186006, 1:1,000, Abcam) or β‐actin (ab8227, 1:2,000, Abcam) overnight at 4°C. The next morning, membranes were washed with TBST three times and probed with horseradish peroxidase‐conjugated (HRP) goat anti‐rabbit IgG H & L secondary antibody (ab6721, 1:10,000, Abcam) for 1 hour at 37°C. After being washed with TBST, immunoreactive bands were developed by enhanced chemiluminescence (Beyotime Institute of Biotechnology) and the grey values of each blot were calculated by ImageJ version 1.50 (National Institutes of Health). The relative expression of the target protein was calculated by normalization to β‐actin.

hDPCs were lysed using RIPA lysis buffer. Total protein was measured using a BCA Protein Assay Kit (Beyotime, Haimen, China). Thirty micrograms of protein was separated by electrophoresis on 8% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) with transfer buffer containing 10% methanol. After blocking with TBST containing 5% nonfat milk at room temperature for 1 h, the membranes were probed overnight at 4°C with the primary antibodies (1:2000) including IKKα/β, p-IKKα/β, IκBα, p-IκBα, p65, p-p65, p38, p-p38, ERK1/2, p-ERK1/2, JNK, p-JNK (CST, USA) and GAPDH (Abcam, UK). Subsequently, the membranes were incubated for 1 h with secondary antibodies at a dilution of 1:2000 (CST, USA) at room temperature. After the membranes were thoroughly washed with TBST buffer, bands with target proteins were visualized with enhanced chemiluminescence reagents (Millipore, USA) and observed using an ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences, USA). The blots were quantified and normalized using the ImageJ 1.47 software program (National Institutes of Health, MD, USA).