Anti cdk2

Antibodies and reagents used in the study are listed as following: anti-Plk1 (Sigma-Aldrich, P5998, 1:10,000), anti-Flag (Sigma, F3165, 1:5000), anti-HA (Sigma, H9658, 1:5000), anti-G6PD (Protein-tech, 25413-1-AP, 1:1000), anti-G6PD (Abcam, ab993, 1:1000), anti-glutathione S-transferase (anti-GST; Protein-tech, 10000-0-AP, 1:5000), anti-phospho-H3Ser10 (Millipore, 06-570, 1:1000), anti-cyclin B1 (Protein-tech, 55004-1-AP, 1:1000), anti-phospho-threonine (Millipore, AB1607, 1:1000), anti-PGLS (Santa Cruz, SC-398833, 1:1000), anti-6PGD (Protein-tech, 14718-1-AP, 1:1000), anti-CDK2 (Protein-tech, 10122-1-AP, 1:1000), rabbit IgG (Protein-tech), mouse IgG (Santa Cruz), and anti-Actin (Protein-tech, 60008-1-Ig, 1:5000). NADP⁺, NADPH, GSH, G6P, 6PG, NAC, Dox, tetracycline, HU, propidium iodide, nocodazole, 4,6-diamidino-2-phenylindole (DAPI), casein, D₂O, ribonucleosides, and deoxyribonucleosides were purchased from Sigma-Aldrich; R5P, nucleotides (AXP, GXP, CXP, and UXP, X = M, D, and T) were purchased from Sangon Biotech. BI2536 was purchased from Selleck Corporation, PP2A was from Millipore, and human recombinant Plk1 was from Sino biological company. [2-¹³C]-glucose and [U-¹³C]-glucose were from Cambridge Isotope Laboratories. γ-³²P ATP was from China Isotope & Radiation Corporation.

Western blot analysis to assess protein expression was performed as previously described [18 ]. The antibodies against E2F1, E2F2 and β-ACTIN were purchased from Cell Signaling, and anti-CDK2, -CDK4, -P16, and -P21 antibodies were purchased from Proteintech. The cells were washed in phosphate-buffered saline (PBS), and proteins was extracted in RIPA buffer. Lysates were cleared by centrifugation, and protein concentrations were estimated using the Bio-Rad protein assay (Bio-Rad, Milan, Italy). Then, 50 μg of protein/lane was loaded onto an acrylamide gel and separated by SDS–PAGE under denaturing conditions. The separated proteins were then transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane soaked in transfer buffer. Non-specific binding was blocked by incubating the blots in 5% non-fat dry milk in PBST for 60 min. After washing, the blots were incubated overnight at 4°C with the primary antibody and β-ACTIN was used as a reference protein. After incubation with the primary antibodies and washing in PBST, anti-mouse or anti-rabbit secondary antibody (both diluted 1:5000) was added (as appropriate) and incubated for 2 h at room temperature. In each experiment, the same amount of protein was used, and each experiment was repeated independently at least three times.

The cells were prepared in RIPA buffer (Solarbio, #R0020). A BCA™ Protein Assay kit (Applygen, #P1511) was used to determine the protein concentration. The proteins (40 μg/sample) were separated by different polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, #IPVH00010), and incubated with the corresponding primary antibody at 4°C overnight. Then, the membranes were incubated with the corresponding secondary antibodies at room temperature for 1 h and measured with a chemiluminescence reagent ECL kit (Advansia, #K-12045-D50).
The primary antibodies used in the experiment used were: anti-PRPS1 (Proteintech, #15549-1-AP), anti-cyclin E1 (Proteintech, 11554-1-AP), anti-CDK2 (Proteintech, 10122-1-AP), anti-P16 (Proteintech, #10883-1-AP), anti-Bax (Proteintech, 50599-2-Ig), anti-Bcl2 (Proteintech, 12789-1-AP), anti-Cleaved-caspeas3 (CST, #9664), anti-MMP2 (Abcam, ab37150), anti-MMP9 (Abcam, ab76003), anti-MMP13 (Proteintech, 18165-1-AP), anti-E-Cadherin (Proteintech, 20874-1-AP), anti-N-Cadherin (Proteintech, 22018-1-AP), anti-Vimentin (Proteintech, 10366-1-AP), anti-NRF2 (Abcam, ab89443), anti-β-actin (Bioss, bs-0061R), and Tubulin (Abcam, #ab7291). The secondary antibodies used in the experiment were anti-rabbit IgG (Abcam, #ab6721) and anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-035-003).

The proteins were extracted from ccRCC cells and HK-2 cells with RIPA buffer (Beyotime Institute of Biotechnology, China) supplemented with 1% protease and phosphatase. The total protein concentration was subsequently detected with a BCA kit (Thermo, Germany). The proteins (20 μg) were subjected to SDS–PAGE and transferred onto polyvinylidene fluoride membranes (FVDF, 0.45 μm; Merck Millipore). After being blocked with 5% skim milk for 1 h and incubated with anti-SGOL1 (Proteintech, 16,977–1-AP), anti-P21 (Proteintech, 10,355–1-AP), anti-cyclin D1 (Proteintech, 26,939–1-AP), anti-CDK2 (Proteintech, 10,122–1-AP), anti-β-actin (Proteintech, 20,536–1-AP), anti-MMP2 (CST, 40,994), anti-MMP9 (CST, 13,667), anti-cyclin E1 (CST, 20,808), anti-E-cadherin (CST, 3195), and anti-N-cadherin (CST, 13,116) antibodies at 4°C overnight, the PVDF bands were incubated with secondary antibodies and detected by enhanced ECL chemiluminescent assay and visualized by Bio-Rad software after washing with TBST three times.

All human GC cell lines (BGC823, HGC27, MGC803, MKN45, and SGC7901), normal gastric cell line (GES-1) and human embryonic renal cell line 293FT were obtained from the American Type Culture Collection (ATCC, Beijing, China). All cell lines were tested mycoplasma-negative. MG132 and CHX were obtained from Sigma (Shanghai, China). Anti-ARIH2, anti-p21, anti-p27, anti-CDK1, anti-CDK2, anti-α-Tubulin, anti-HA, anti-SKP2, anti-RNF126 and anti-UHRF2 antibodies were purchased from Proteintech (Wuhan, China). Anti-MYC, anti-Flag, anti-phospho-ATR, anti-phospho-ATM, anti-ɣ-H2AX and anti-cleaved-caspase-3 antibodies were obtained from Cell Signaling Technology (Shanghai, China). Anti-Ki67 and anti-Bcl2 antibodies were purchased from BD Biosciences. All antibodies were diluted according to the manufacturer’s instructions.

Total proteins were collected at 48 h after transfection and then electrophoresed using 10–12% SDS-PAGE gels, transferred onto polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA), and blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20. Membranes were incubated overnight at 4°C with primary antibodies followed by the appropriate secondary antibody for 1 h at room temperature. The signals were detected with electrogenerated chemiluminescence (ECL) developer solution (Millipore). Primary antibodies were presented as follows: anti-β-actin (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-xL (1 : 1000; CST), anti-BTG3 (1 : 500; Sigma), anti-cyclinE1 (1 : 1000, CST), anti-P27 (1 : 1000, Proteintech, Rosemont, IL, USA), and anti-CDK2 (1 : 1000, Proteintech). The relative optical density of bands was quantified 36 with a GelPro Analyzer (Media Cybernetics, Silver Spring, MD, USA).

Western blotting method was used in the following experiment [14 (link), 15 ], loading 25 μg of total protein lysate per lane. The following primary antibodies were used: anti-HDAC7 (1:1000, #33,418, CST), anti-c-Myc (1:1000, #5605, CST), anti-CDK1 (1:1000, 19,532-1-AP, Proteintech), anti-Cyclin B1 (1:1000, 55,004-1-AP, Proteintech), anti-CDK2 (1:1000, 10,122-1-AP, Proteintech), anti-CyclinA2 (1:1000, 18,202-1-AP, Proteintech), anti-E-cadherin (1:1000,#14,472, CST), anti-Zeb1 (1:1000, 21,544-1-AP, Proteintech), anti-α-SMA (1:1000, 14,395-1-AP, Proteintech) and anti-β-actin (1:1000, #3700, CST). The secondary antibodies used were HRP-linked anti-IgG antibodies (1:5000, Zhongshan Company, Beijing, China).