Bca protein quantitative kit

Briefly, protein was isolated from the cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer and then quantitated using a BCA protein quantitative kit (Thermo Fisher Scientific). Equal amounts of protein samples (25 μg) were resolved by 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore). After being blocked with milk in Tris-buffered saline-Tween buffer, the membrane was probed with primary antibody specific for FGFR2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling Technology), or phospho-P38 (Cell Signaling Technology), followed by secondary antibodies. The signal was detected using chemiluminescence (Thermo Fisher Scientific). The antibody specific for β-actin (Sigma-Aldrich) was applied to normalize the protein loaded. Densitometric analysis of the bands was performed using the ImageJ software.

Protein expression in LUAD cells was measured by western blot analysis. Briefly, cells were lysed with radioimmunoprecipitation assay (RIPA) protein lysis buffer containing protease inhibitors (cOmplete
^TM Protease Inhibitor Cocktail; Sigma-Aldrich) and phosphorylase inhibitor (Sigma-Aldrich). The protein concentration was quantified using the BCA Protein Quantitative kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For western blot analysis, 20 μg of total protein per sample was separated by 4% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Billerica, USA). The membrane was blocked with 5% nonfat milk buffer for 30 min at room temperature. Then, the primary antibodies, including anti-Vimentin (1:1000, ab8978; Abcam, Cambridge, UK), anti-N-cadherin (1:1000, ab18203; Abcam), anti-E-cadherin (1:1000; Abcam), and anti-GAPDH (1:5000; Abcam), anti-ALNL (1:1000, ab211872; Abcam), anti-RRM2 (1:1000, ab172476; Abcam) were incubated with the membranes at 4°C overnight. After wash with 1× PBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (KS001, KS002; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Finally, the targeted bands were detected using an enhanced chemiluminescence reagent (Millipore) and photographed and quantified using an imaging system (Thermo Fisher Scientific)

After specific treatment, total cellular proteins from K562 cells were extracted by using RIPA Buffer (Auragene, Changsha, China). A BCA protein quantitative kit (Thermo, USA) was used to measure the concentration of protein samples. Equal amounts of protein samples were resolved by 12% SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with primary antibodies (anti-GAPDH; anti-Bax; anti-Bcl-2; anti-Fas; anti-p53; dilution for all, 1: 1000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Subsequently, membranes were incubated with an HRP-conjugated secondary antibody (Anti-rabbit IgG, HRP-linked antibody; 1: 5000) at room temperature for 2 h. To visualize the protein blots, an ECL kit (Applygen, Beijing, China) was used according to the manufacturer’s protocol.

The TRIzol^® kit, BCA protein quantitative kit, SYBR Green PCR kit and reverse transcription kit used in the study were bought from the Thermo Fisher Scientific, Inc. The ECL kit (cat. no. WBKLS0100) was purchased from EDM Millipore. The primary antibodies for PPM1A and phosphorylated (p-)Smad2/3 were purchased from Abcam. The primary antibodies for Smad2/3, p-Smad2/3, E-cadherin, vimentin, α-SMA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology, Inc. PLKO.1, psPAX2 and pMD2G were acquired from Addgene, Inc. DH5α competent cells were purchased from Beijing Transgen Biotech Co., Ltd.; 293T cells, from ATCC and pLVX-Puro from Clontech Laboratories, Inc.