Bca protein assay kit

Cells were lysed in RIPA buffer (CW2333S, CWBIO, China) supplemented with protease inhibitor cocktail set 1 (539131, Millipore, USA) and phosphatase inhibitor. Protein concentrations were measured using BCA protein assay kits (CW0014S, CWBIO). Equal amounts of total cell lysate were separated using 10% SDS-PAEG (P0014B, CWBIO) and transferred to PVDF membranes (ISEQ00010, Millipore, USA) blocked with 5% bovine serum albumin (0332, Ameresco) for 1 h at room temperature. Then the blots were incubated with the primary antibodies against SHMT2 (1:1,000, GTX125939, GeneTex), E-cadherin (1:1,000, 24E10, Cell Signaling), ZO1 (1:1000, D7D12, Cell Signaling), N-cadherin (1:1,000, D4R1H, Cell Signaling), Vimentin (1:1,000,D21H3,Cell Signaling, β-catenin (1:1,000, D10A8, Cell Signaling), p21^Cip1 (1:1000, 12D1, Cell Signaling), p27^Kip1 (1:1000, D69C12, Cell Signaling), Cyclin D1(1:1000, E3P5S, Cell Signaling), CDK4(1:1000, D9G3E, Cell Signaling), β-actin (1:1000,13E5, Cell Signaling), and GAPAH (1:1,000,D16H11, Cell Signaling) at 4 ℃ overnight. Membranes were incubated in HRP-conjugated secondary antibody (1:2000, 7074S, Cell Signaling) for 1 h at room temperature. The protein bands were exposed using a chemiluminescent substrate (WBKLS0500, Millipore, USA), and quantitatively analyzed using ImageJ [19 (link)].

Briefly, the cells were rinsed with sterile PBS and then lyzed with RIPA lysis buffer (AR0105; Boster, Wuhan, China) containing protease and phosphatase inhibitors. Then, the protein concentration was analyzed using a BCA protein assay kit (CW0014; CWBIO, Taizhou, China). Then, equivalent amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. Then, the membranes were blocked and incubated with the specified primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies. Finally, the levels of protein expression were detected using chemiluminescent reagents (WBKLS0500; Millipore).

Bladder cancer cells were washed with phosphate-buffered saline and lysed in RIPA buffer (cwbiotech) containing protease inhibitor. The protein concentration was quantified using the BCA Protein Assay Kit (cwbiotech). For each sample, an amount of 20 μg total protein was separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes for 1 h and a half at 320 mA in transfer buffer. After blocking with 5% degreased milk at room temperature for 1 h, the membranes were incubated overnight at 4 °C with rabbit polyclonal anti-human LBHD1 (1:1000, Abcam) and mouse monoclonal anti-GAPDH (1: 2,000, cwbiotech). After washing, the membranes were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Immunoreactive bands were incubated with ECL western blotting detection reagents (Amersham Biosciences) then visualized using a western blotting detection system (ECL Plus; Amersham Pharmacia Biotech, Little Chalfont, UK). The film was scanned, and the intensity of the bands was quantified by densitometry (Image J 1.47 software) and normalized to the corresponding GAPDH bands. All of the Western immunoblots were performed at least three times.

Cells were harvested and lysed in radioimmunoprecipitation buffer (Beyotime, China). Quantification was performed using the BCA protein assay kit (CWBIO, China). Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), after blocking with 2% skim milk or bovine serum albumin (BSA). The membrane was then treated with primary antibodies at 4°C overnight and then washed using 1× Tris-Bufferd Saline and Tween 20 (TBST, CWBIO, China). In the end the membranes were incubated with secondary antibodies (EMAR, China) for 1 h at room temperature. The protein expression level was measured by Immobilon ECL Ultra Western HRP Substrate (Millipore, USA) using GeneGnome XRQ system (Syngene, USA).