Protease inhibitor phenylmethanesulfonyl fluoride

The cells were lysed with RIPA buffer (Beyotime, shanghai, China) and 1 mM protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, shanghai, China), agitated for 15 minutes at 4℃, and centrifuged at 12,000 rpm for 25 minutes. Protein concentrations were measured with the BCA Protein Assay (Beyotime, Shanghai, China). Proteins were electrophoresed on SDS-polyacrylamide gels and transferred onto a polyvinylidene fluoride for analysis. Then, the membrane was incubated with rabbit anti-PAX2 (1:200 dilutions; Invitrogen, Carlsbad, CA), anti-CDK1(1:2000 dilutions; Abcam, San Francisco, USA) and anti-GAPDH antibody(1:5000 dilution; Abcam, San Francisco, USA) overnight at 4℃, respectively, and then incubated in the goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilution; MT-bio, shanghai, China) at room temperature for 1 hour. Antibody binding was visualized using ECL chemiluminescence reagent (Thermo, MA, USA). GAPDH was used as an internal control to normalize other proteins expression level.

GAPDH and CDC25A rabbit anti-human antibodies were purchased from Cell Signaling Technology. CCNB1 rabbit anti-human antibodies were bought from Abcam. Anti-rabbit antibodies for GAPDH, CDC25A, and CCNB1 and secondary antibodies of IRDye 800 raised in goat were purchased from Li-Cor Biosciences (Lincoln, NE, USA). The proteins were extracted from cells using Western blot and IP kits (Beyotime, Beijing, China) and protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, Beijing, China). Protein concentration was detected by enhanced BCA protein assay kit (Beyotime, Beijing, China). The Western blot procedure was performed as described in our previous publication.14 (link) The membranes of the protein blots were scanned by Odyssey Software Version 3.0 system (Li-Cor Biosciences Lincoln, NE, USA). GAPDH protein was used as an internal reference to calculate the expression of each protein.

Cells were lysed with lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitor phenylmethane sulfonyl fluoride (Beyotime Institute Biotechnology), and the supernatant of the lysed cells was recovered. Protein concentrations were quantified using a bicinchoninic acid protein assay. Protein samples (50 µg per lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Non-specific binding was blocked with 5% skim milk in TBS containing 0.1% Tween-20 for 1 h at room temperature. Subsequently, blotted membranes were incubated overnight at 4°C with specific primary antibodies. The following primary antibodies were used: Monoclonal rabbit anti-human podoplanin antibody (1:1,000; cat. no. 9047; Cell Signaling Technology, Inc., Danvers, MA, USA); monoclonal mouse anti-human FGF1 antibody (1:500; cat. no. H00002246-M02; Abnova Corporation, Taipei, Taiwan); and monoclonal mouse anti-human α-tubulin antibody (1:5,000; cat. no. 66031-1-Ig; ProteinTech Group, Inc., Chicago, IL, USA) as a loading control. Detection was performed using horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. nos. SA00001-1 and SA00001-2; ProteinTech Group, Inc.). Finally, blots were immersed in ECL detection reagent (EMD Millipore, Billerica, MA, USA) and exposed to a ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc.).