The following were purchased from the sources shown: reagents and culture media (Thermo Fisher Scientific Inc.), 1:1000‐diluted anti‐β‐actin (sc‐47 778; Santa Cruz Biotechnology Inc.), 1:1000‐diluted anti‐Flag (A8592; Sigma‐Aldrich Corp.), and 1:500‐diluted anti‐CPVL (ab180147), 1:500‐diluted anti‐p‐Rb (ab184796), 1:500‐diluted anti‐PTEN (ab267787), and 1:500‐diluted anti‐cyclin E1 (ab33911; all from Abcam).
Ab184796
Manufactured by Abcam
Sourced in United Kingdom
Ab184796 is a polyclonal antibody specifically targeting Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). It is a protein-based reagent commonly used in various life science applications to detect and quantify GAPDH expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ab181616, by Abcam (2 mentions)
Ab134175, by Abcam (2 mentions)
Anti cyclin e1, by Abcam (1 mentions)
Ab180147, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Bca protein quantification kit, by Vazyme (1 mentions)
Ab192239, by Abcam (1 mentions)
Ecl kit, by Vazyme (1 mentions)
Ab71535, by Abcam (1 mentions)
Hydrogen peroxide h2o2, by Merck Group (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Ab189494, by Abcam (1 mentions)
Ab81299, by Abcam (1 mentions)
Ab68266, by Abcam (1 mentions)
2 hydroxypropyl β cyclodextrin solution, by Merck Group (1 mentions)
Ab211542, by Abcam (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab199728, by Abcam (1 mentions)
Ab54576, by Abcam (1 mentions)
7 protocols using ab184796
1
Immunoblotting Assay for Cellular Proteins
2
Protein Extraction and Western Blot Analysis
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The RIPA buffer was used to extract the total protein (Solarbio, Beijing, China). Protein quantification was done with the BCA Protein Quantification Kit (Vazyme). After that, following electrophoresis by SDS-PAGE, the separated proteins (50 µg) were electroblotted from the gel on PVDF membranes (Sigma-Aldrich). followed by an overnight treatment at 4 °C with primary antibodies: CDKN2C (ab192239, Abcam, Cambridge, USA), Cyclin D1 (60186-1-Ig, Proteintech, Wuhan, China), RB1 (ab181616, Abcam), p-RB1 (ab184796, Abcam), CDK4 (11026-1-AP, Proteintech), Bcl-2 (12789-1-AP, Proteintech), BAX (50599-2-Ig, Proteintech), and then 1.5-h interaction with secondary antibody (Abcam) at room temperature (RT). Chemiluminescence was seen with the ECL kit (Vazyme).
3
Xenograft Tumor Immunohistochemistry Analysis
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The xenograft tumor tissues were dissected and fixed with 10% phosphate-buffered neutral formalin overnight prior to paraffin embedding. The paraffin blocks were then cut into sections and incubated with rabbit anti-human cyclin D1 (ab134175), cyclin E1 (ab71535), p-Rb (ab184796) (Abcam, Cambridge, UK), and E-Cadherin (AF0138) and Vimentin (AF0318) antibodies (Beyotime Institute of Biotechnology) (all diluted 1:500).
4
Senescence Induction and Characterization
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The hydrogen peroxide (H2O2) was from Sigma-Aldrich (St. Louis, MO, USA). The 7-KC was from Sigma-Aldrich and dissolved in 45% (w/v) 2-hydroxypropyl-β-cyclodextrin solution (Sigma-Aldrich). Type-V collagenase (C9263) was fromSigma-Aldrich (St. Louis, MO, USA). SA-β-gal activity kit was from GenMed Scientific (Shanghai, China). Cell cycle and apoptosis analysis kit was from Beyotime (Shanghai, China). The antibodies against CDKN2A/p16INK4a (ab211542), γ-H2A.X (ab81299), p21 (ab88224), SIRT1 (ab189494), CDK4 (ab68266), and phosphorylated retinoblastoma (p-Rb) protein (ab184796) were from Abcam (Cambridge, UK). The antibodies against E2F1 (A19579), insulin (a19066) were from ABclonal (Wuhan, China). The anti-p53 antibody (10442–1-AP) was from Proteintech (Wuhan, China). The anti-p-p38MAPK antibody (4511) was from Cell Signaling Technology (Boston, MA, USA). The anti-β-gal antibody (A11132) was from Thermo Fisher Scientific (Waltham, MA, USA).
5
Western Blot Analysis of Cell Cycle Regulators
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Cells were resuspended in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Sodium deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4). Cell lysates were incubated on ice for 20 mins and supernatants were collected after high-speed centrifuge at 4°C. Protein concentration was measured by Bradford assay. Samples were loaded evenly into SDS-PAGE and transferred onto PDVF membranes (Roche). Membranes were blocked in 5% skim milk at room temperature and then incubated with primary antibodies overnight at 4°C. After washing with PBS-T (PBS solution, 0.1% Tween-20) and incubation of secondary antibodies, membranes were incubated with ECL Western Blotting Substrate (Thermo Fisher Scientific) and protein bands were developed in dark room. Antibodies used for Western blot assay were: CCND1 (abcam, ab16663), pRb (abcam, ab184796), CDK4 (abcam, ab199728), CDK6 (abcam, ab54576), GAPDH (abcam, ab9485).
6
Western Blotting and Immunofluorescence for Vascular Smooth Muscle Cell Analysis
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For Western blotting, protein concentrations in VSMC lysates were quantitated using a BCA Protein Assay Kit (Thermo Fisher Scientific, No. 23225). Proteins (30 μg) were separated through 15% SDS/PAGE and then transferred on to PVDF membranes (Millipore). After 1 h incubation in 5% non-fat milk at room temperature, the blots were further incubated with primary antibodies overnight at 4°C. The primary antibodies used were: anti-Cyclin D1 (Abcam, ab134175), anti-Cyclin E (Abcam, ab33911), anti-cyclin-dependent kinase (CDK) 2 (CDK2) (Abcam, ab32147), anti-CDK4 (Abcam, ab108357), anti-Rb (phospho S807) (Abcam, ab184796), anti-β actin (HC-101-02), anti-active caspase3 (Abcam, ab2302), anti-cleaved poly ADP-ribose polymerase (PARP) (Abcam, ab4830), anti-α-SMA (Abcam, ab32575), anti-SM22α (Abcam, ab14106), anti-Calponin (Abcam, ab46794), anti-Osteopontin (Abcam, ab8448), anti-smoothelin (Abcam, ab21108), and anti-tropomyosin (Abcam, ab181085).
For immunofluorescence, VSMCs were incubated in primary antibodies against α-SMA (Abcam, ab32575) or calponin (Abcam, ab46794).
For immunofluorescence, VSMCs were incubated in primary antibodies against α-SMA (Abcam, ab32575) or calponin (Abcam, ab46794).
7
Quantitative gene expression and protein analysis
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Quantitative real-time RT-PCR (qRT-PCR) and immunoblot analyses were performed following procedures as described previously [59 (link), 60 (link)]. Relative mRNA levels were determined using the comparative 2−ΔΔCT method and normalized against β-actin. The specificity of the primers was validated by the melting-curve detection. PCR cycle times ≥ 34 were considered to be below detection. For quantitative methylation-specific PCR (qMSP), bisulfite conversion method was used (EZ DNA Methylation-Gold Kits, Zymo Research), with the type II collagen gene (COL2A1) used as the internal reference gene. Primer sequence information is listed in the Supplementary Table S1 . For immunoblot analysis, a chemiluminescence method was used for immunosignal detection (Amersham ECL Western Blotting Detection System). Primary antibodies used are as follows: HNF4α (K9218, abcam), p21Waf1/Cip1 (12D1, Cell Signaling Technology), β-actin (C4, Santa Cruz Biotechnology), FLAG (M2, Sigma-Aldrich), p53 (sc-126, Santa Cruz Biotechnology), Rb (ab181616, abcam), and phospho-Rb (ab184796, abcam).
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