Total protein from tissues or cells was initially isolated using a high-efficiency radioimmunoprecipitation assay lysis buffer (R0010, Solarbio, Beijing, China). Thereafter, the concentration of the extracted protein was estimated with a bicinchoninic acid kit (20201ES76, YEASEN, Shanghai, China). After separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was blotted onto polyvinylidene fluoride membranes. Following 1-h blocking at ambient temperature using 5% bovine serum albumin, the membranes were probed overnight at 4°C with primary rabbit antihuman antibodies (Abcam, Cambridge, UK) to PDCD4 (ab51495, 1: 100), PD-L1 (ab205921, 1: 50), PI3K (ab32089, 1: 100), phosphorylated (p)-PI3K (ab182651, 1: 100), Akt (ab8805, 1: 50), and p-Akt (ab81283, 1: 500). The following day, the membranes were reprobed with goat antirabbit immunoglobulin G (IgG) (ab205718, 1: 1000, Abcam) labeled by horseradish peroxidase (HRP) at ambient temperature for 1 h. The immunocomplexes on the membrane were developed with VILBER Fusion FX5 (Vilber Lourmat, Limoges, France), followed by the quantification of band intensities using the ImageJ 1.48u software (National Institutes of Health, Bethesda, MA). The relative protein expression was represented by the ratio of the gray value of the target band to that of GAPDH.
Ab51495
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab51495 is a laboratory equipment product. It is designed for use in scientific research and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ab8245, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bicinchoninic acid kit, by Yeasen (1 mentions)
Fusion fx5, by Vilber (1 mentions)
Primary rabbit antihuman antibodies, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Anti β catenin rabbit polyclonal antibody, by Cell Signaling Technology (1 mentions)
Benchmark xt system, by Roche (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab222772, by Abcam (1 mentions)
Ab92494, by Abcam (1 mentions)
Ab181616, by Abcam (1 mentions)
Ab126785, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab32147, by Abcam (1 mentions)
7 protocols using ab51495
1
Quantitative Analysis of Protein Expression
2
Western Blot Analysis of PDCD4 Protein
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The total protein of transfected cells were obtained by radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China), and then the protein samples were subsequently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then the proteins were accordingly transferred onto a polyvinylidene fluoride membrane (Millipore), which were then blocked with 5% skimmed milk for 1 h at ambient temperature. Notably, the membranes were specifically incubated with rabbit anti-PDCD4 antibody (1:1000, ab51495, Abcam, Cambridge, UK) or mouse anti-GAPDH antibody (1:2000, ab8245, Abcam, Cambridge, UK) at 4° C overnight, and then with HRP-conjugated secondary antibodies at ambient temperature for 1 h. Ultimately, the protein bands were developed by an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).
3
Immunohistochemical Analysis of Colon Lesions
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We used paraffin-embedded sections of the colons of the mice in all groups for the immunohistochemical analysis. Serial histological sections (4 μm thickness) were made from each paraffin wax block. Immunohistochemical staining was performed automatically (Ventana Benchmark XTsystem; Ventana, Touchstone, Arizona, USA), according to the manufacturer's instructions. The primary antibodies were anti-MCM2 rabbit monoclonal antibody (no. 3619, anti-MCM2 (D7611)XP, 1:400 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-β-catenin rabbit polyclonal antibody (#9661, 1:200 dilution; Cell Signaling Technology) and anti-PCDC4 rabbit polyclonal antibody (ab51495, 1:500 dilution; Abcam, Inc. Cambridge, MA, USA). In each case, the positive and negative controls were run concurrently. As the final step, the sections were lightly counterstained with Mayer's hematoxylin (Merck, Tokyo, Japan).
Immunoreactivity for antibodies against MCM2, PDCD4 and β-catenin was assessed in the lesions (high-grade dysplasia) that developed in groups 1 through 3 using a microscope (Olympus BX41, Olympus Optical Co., Tokyo, Japan). The intensity and localization of the immunoreactivity against the primary antibodies were determined by a pathologist (T.T.) who was unaware of the treatment group to which the slide belonged.
Immunoreactivity for antibodies against MCM2, PDCD4 and β-catenin was assessed in the lesions (high-grade dysplasia) that developed in groups 1 through 3 using a microscope (Olympus BX41, Olympus Optical Co., Tokyo, Japan). The intensity and localization of the immunoreactivity against the primary antibodies were determined by a pathologist (T.T.) who was unaware of the treatment group to which the slide belonged.
4
Western Blot Analysis of Protein Expression
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Following transfection, cells and tissues were lysed for 48 h using RIPA Lysis and Extraction Buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and the protein concentration was measured using Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Following heating at 100°C for 10 min in the presence of a loading buffer, equal amounts of protein lysates (50 µg) were separated using 10% SDS-PAGE (Bio-West Inc., Logan, UT, USA) at 100 V for 1 h, and transferred onto Invitrogen nitrocellulose membranes (Thermo Fisher Scientific, Inc.) at 120 V for 1 h. Following blocking with 5% skimmed milk (diluted with PBS), the membranes were incubated overnight at 4°C with the following primary antibodies: CYP27A1 (ab126785; 1:500; Abcam, Cambridge, MA, USA), c-myc (ab32072; 1:500; Abcam), RB (ab181616; 1:500; Abcam), Ki-67 (ab15580; 1:500; Abcam), CDK2 (ab32147; 1:500; Abcam), p21 (ab109520; 1:500; Abcam), p53 (ab32049; 1:500; Abcam), PDCD4 (ab51495; 1:500; Abcam), SOX2 (ab92494; 1:500; Abcam), β-actin (ab8227; 1:500; Abcam). Subsequently, the membranes were incubated with secondary goat monoclonal (RMG01) to rabbit IgG Fab region (Biotinylated; ab222772; 1:10,000; Abcam) at room temperature for 2 h, and proteins were detected using enhanced chemiluminescence (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).
5
Quantification of PDCD4 Protein Levels
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The cellular protein was extracted using RIPA lysis buffer supplemented with proteinase inhibitor (KeyGen, Nanjing, China). Briefly, 20 μg of protein samples were separated using SDS/PAGE and then transferred on to PVDF membranes (Millipore, Billerica, MA, U.S.A.). The PVDF membranes were incubated with primary antibodies overnight at 4°C, including anti-PDCD4 (ab51495; Abcam, Cambridge, MA, U.S.A.) and anti-β-actin (ab8224; Abcam). Subsequently, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C. The bands were developed using ECL Western Blotting Detection Kit (Thermo Fisher, Waltham, MA, U.S.A.).
6
Protein Extraction and Western Blot Analysis
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Proteins from cells and the coronary artery tissues were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Beijing, China). The protein was quantified by BCA protein assay, and 25 μg proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, USA). After being blocked with 5% non-fat milk for 1 h, the membranes were incubated with the primary antibodies overnight at 4°C. The primary antibodies used in this study were as following: anti-WWP2 (1:5000; ab103527, Abcam), anti-ICAM-1 (1:1000; #4915, Cell Signaling Technology, Beverly, USA), anti-VCAM-1 (1:1000; #13662, Cell Signaling Technology), anti-PDCD4 (1:5000; ab51495, Abcam), anti-HO-1 (1:2000; ab13243, Abcam), anti-p47 (1:1000; #4312, Cell Signaling Technology), anti-HA (1:1000; 3724S, Cell Signaling Technology), anti-Myc (1:1000; 2276S, Cell Signaling Technology), anti-Flag (1:1000; 14793S, Cell Signaling Technology) and GAPDH (1:5000; ab8245, Abcam). Next, the membranes were incubated with HRP-labeled secondary antibodies at room temperature for 2 h. Then, the protein blots were visualized by using ECL kit (Thermo Fisher Scientific), and the gray level of bands were analyzed by using Image J software (NIH, Bethesda, USA). The relative protein levels were normalized to GAPDH.
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted by RIPA lysis buffer (Beyotime, Shanghai, China). Subsequently, protein concentration was measured using a bicinchoninic assay. Then 30 μg of protein / lane was separated via 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore, Schwalbach, Germany). After being blocked with TBS containing 5% skimmed milk at room temperature for 1 h, the membrane was incubated with rabbit anti-PDCD4 antibody (1:1000, ab51495, Abcam, Cambridge, UK) and anti-GAPDH antibody (1:2000; ab37168, Abcam, Cambridge, UK), respectively at 4 °C overnight, and then incubated with HRP-conjugated secondary antibodies (1:5000, Beyotime, Shanghai, China) at room temperature for 1 h. GAPDH was used as the internal control. Ultimately, the protein bands were developed by the enhanced chemiluminescence reagent (Beyotime, Shanghai, China).
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