Anti cyclin e1

Total protein was extracted from the samples, as previously described.20 (link)–22 (link) Primary antibodies used were anti-LRP11, anti-P16, anti-CDK 2 (1:1000, Abcam, ab 32147), anti-CDK 4 (1:1000, Cell Signaling Technology, Danvers, MA, USA, #12790), anti-Cyclin D1 (1:1000, Cell Signaling Technology, Danvers, MA, USA, #2978), anti-cyclin E1 (1:1000, Abcam, ab 3927), anti-cleaved-PARP (1:1000, Cell Signaling Technology, Danvers, MA, USA, #5652T), anti-BcL-xL (1:200, Santa Cruz Biotech, sc-8392), anti-Bax (1:200, Santa Cruz Biotech, sc-7480), anti-MMP-2 (1:1000, Abcam, ab 92536), anti-MMP-9 (1:1000, Cell Signaling Technology, Danvers, MA, USA, #13667), anti-VEGF (1 μg/mL, Abcam, ab 46154), and anti-β-actin (1:1000, Cell Signaling Technology, #4970). β-actin is currently recognized as very stable internal control. For mammalian cell expression, it refers to a protein encoded by the housekeeping gene. Secondary antibodies were anti-rabbit and anti-mouse IgG peroxidase conjugates (1:5000, Merck Millipore, MA, USA), as well as anti-sheep IgG cell and tissue staining kit (R&D Systems, USA, CTS019). The ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Logan, UT, USA) was used to detect these proteins, and the results were analyzed by ImageJ software (NIH, Bethesda, MD, USA).

Total protein was isolated from cell lysates by using RIPA buffer, and quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and then transferred onto PVDF (Bio-Rad) membranes. After blocking with 5% skim milk in TBST for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study were as follows: anti-p27 Kip1 (Abcam, Cambridge, MA, USA; 1:1000), anti-Bax (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Bcl-2 (Abcam; 1:1000), anti-cleaved caspase 9 (Abcam; 1:1000), anti-CDK2 (Abcam; 1:1000), anti-Cyclin E1 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.

Western blotting was conducted as previously described [17 (link)] using anti-HIF-1α (1:1000; Cell Signaling Technology, Danvers, MA, United States), anti-cyclin E1 (1:1000; Abcam, Cambridge, United Kingdom), anti-cyclin A2 (1:1000; Abcam), anti-CDK2 (1:1000; Abcam), anti-p21 (1:1000; Abcam), anti-CD99 (1:1000; Abcam), anti-ERK1+ERK2 (1:5000; Abcam), anti-ERK1 (phospho T202)+ERK2 (phospho T185) (1:1000; Abcam) and anti-GAPDH (1:1000; Sangon Biotech Corp., Shanghai, China) primary antibodies. The membranes were then incubated with secondary anti-rabbit antibodies conjugated to horseradish peroxidase (1:4,000; Abcam). Proteins were detected using Pierce^TM enhanced chemiluminescence (ECL) western blot analysis substrate (Thermo Fisher Scientific Inc.) and analyzed with ImageJ software (NIH, Bethesda, MD, United States).

The cellular protein was harvested using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Beyotime). The protein concentrations were detected using a BCA kit (Beyotime). The western blot was conducted as previously described[7 (link)]. The primary antibodies were anti-β-actin (Abcam, Cambridge, United Kingdom), anti-GAPDH (Abcam), anti-hypoxia-inducible factor 1α (HIF-1α) (Cell Signaling Technology, Danvers, MA, United States), anti-c-MYC (Abcam), anti-proliferating cell nuclear antigen (Abcam), anti-CDK2 (Abcam), anti-CDK4 (Abcam), anti-CDK6 (Abcam), anti-CyclinD1 (Abcam), anti-CyclinE1 (Abcam), anti-AKT (Abcam), and anti-phospho-AKT (Abcam).

Cells were harvested, lysed, and processed for Western blot analysis as described previously using 150 mM NETN lysis buffer [20 mM tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM phenyl-methyl-sulfonyl fluoride, leupeptin (10 mg/ml), and aprotinin (10 mg/ml)]. For cell fractionation, we isolated cytoplasmic and soluble nuclear fractions with the NE-PER Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol; to isolate the chromatin fraction, the insoluble pellet was resuspended in radioimmunoprecipitation assay (RIPA) buffer and sonicated in a Bioruptor according to the manufacturer’s protocol (medium power, 20 min, 30 s on, 30 s off at 4°C). Proteins were separated using SDS–polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk phosphate-buffered saline (PBS)/Tween and incubated with primary antibody (Ab) for overnight at 4°C. Abs for Western blot analysis included anti–β-actin (Sigma-Aldrich), anti-FANCJ (E67), anti-vinculin (Sigma-Aldrich), anti–cyclin E1 (Abcam), and anti-p21 (BD Pharmingen). Membranes were washed and incubated with horseradish peroxidase–linked secondary Abs (Amersham) for 1 hour at room temperature (RT) and detected by chemiluminescence (Amersham).