The expression of CDKN2C and β-tubulin was assessed by western blot as described5 (link) using a monoclonal rabbit anti-CDKN2C Ab (anti-p18 INK4c, ab192239, Abcam, 1:1000), a rabbit polyclonal anti-β-tubulin Ab (GTX101279, Gentex, 1:3000), and a rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH; ab9485, Abcam, 1:2500), respectively. Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Research 111-035-144, 1:10,000) was used as a secondary Ab. Protein expression was assessed using the ChemiDoc™ Imaging System (BioRad).
Ab192239
Manufactured by Abcam
Sourced in United States
Ab192239 is a polymer detection system used for immunohistochemistry applications. It contains the necessary components for the visualization of target proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (1 mentions)
Gtx101279, by Gentex (1 mentions)
Ab9485, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein quantification kit, by Vazyme (1 mentions)
Ab181616, by Abcam (1 mentions)
Ab184796, by Abcam (1 mentions)
Ecl kit, by Vazyme (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Ab5823, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
Ab124762, by Abcam (1 mentions)
Ab75974, by Abcam (1 mentions)
P0493, by Merck Group (1 mentions)
Ab32072, by Abcam (1 mentions)
Steponeplus apparatus, by Thermo Fisher Scientific (1 mentions)
Thermoscript rt pcr system, by Thermo Fisher Scientific (1 mentions)
Ab108349, by Abcam (1 mentions)
Infinite 200, by Tecan (1 mentions)
6 protocols using ab192239
1
Quantitative Western Blot Analysis
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
Western Blot Analysis of Protein Expression
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Cells were trypsinized, washed with phosphate- buffered saline (PBS) and lysed in Cell lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% Triton X-100, Sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin). Equal amounts of protein were separated by 8 ~ 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Roche). The membranes were blocked for 1 ~ 2 h at room temperature in PBST with 5% (w/v) non-fat milk and incubated overnight at 4°C with primary antibodies. After incubation with secondary antibodies, proteins were visualized with the ECL detection system (Thermo Fisher Scientific). The following antibodies were used: PRMT5 (P0493, Sigma), c-Myc (ab32072, ab56, Abcam), GAPDH (M171-3, MBL), PTEN (ab32199, Abcam), p21 (#2947, CST), p63 (ab124762, Abcam), p18 (ab192239, Abcam), p57 (ab75974, Abcam), HSP70 (#4873, CST), H4R3me2s (ab5823, Abcam) and Histone H4 (16047-1-AP, Proteintech).
4
Validating INK4 Expression in HCC
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Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to confirm the expression of INK4 in HCC and adjacent tissues. Thirty patients diagnosed with HCC were randomly selected from the Second Hospital of Dalian Medical University, and patient information is presented in Supplementary Table S1. RNA was extracted from both HCC and adjacent tissues for cDNA amplification and qRT-PCR using the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA, USA) and StepOnePlus apparatus (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene. The primer sequences for GAPDH and INK4 family members are listed in Supplementary Table S2. Immunohistochemical staining was performed to validate INK4 expression in HCC tissues. The following antibodies were used as primary antibodies: CDKN2A (ab108349, Abcam, Cambridge, UK), CDKN2B (AF0230, Affinity Biosciences, Cincinnati, OH, USA), CDKN2C (ab192239, Abcam), and CDKN2D (10272-2-AP, Proteintech, Rosemont, IL, USA). Goat antimouse Alexa fluor SP-9000 from ZSGB Biotechnology (Beijing, China) was used as a secondary antibody.
5
Quantifying Cytokine and Protein Levels
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IL-6 and TNF-α concentrations were quantified in 24 h culture supernatants by ELISA (eBioscience) following manufacturer’s instructions and analyzed on a multimode spectra microplate reader (Infinite 200, Tecan) with the Magellan™ software (Tecan).
Capillary-based “Simple Western System” (WES, ProteinSimple) was used according to the manufacturer’s protocol to quantify IL1-ß protein, using goat anti mouse IL-1b/ IL-1F2 (AF-401-NA, R&D systems, 1/250), and p18 protein, using rabbit monoclonal anti-p18 (ab192239, Abcam). Protein expression was normalized by ß-actin quantification using rabbit anti-mouse ß-actin (4970, CTS, 1/250) as described previously [15 (link)]. Briefly, protein extracts from 50000 sorted cells were adjusted to 0.15 mg/ml concentration. Separation and immunoprobing were performed automatically and the chemiluminescent signals were detected and analyzed by Compass software (ProteinSimple).
Capillary-based “Simple Western System” (WES, ProteinSimple) was used according to the manufacturer’s protocol to quantify IL1-ß protein, using goat anti mouse IL-1b/ IL-1F2 (AF-401-NA, R&D systems, 1/250), and p18 protein, using rabbit monoclonal anti-p18 (ab192239, Abcam). Protein expression was normalized by ß-actin quantification using rabbit anti-mouse ß-actin (4970, CTS, 1/250) as described previously [15 (link)]. Briefly, protein extracts from 50000 sorted cells were adjusted to 0.15 mg/ml concentration. Separation and immunoprobing were performed automatically and the chemiluminescent signals were detected and analyzed by Compass software (ProteinSimple).
6
Western Blot Protein Detection
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The procedures were performed according to our previous study [47] . The primary antibodies used in western blot were as follows: anti-ACTIN (20536-1-AP, Proteintech), anti-ZFHX3 (PD010, MBL), anti-FTO (27226-1-AP, Proteintech), anti-MYC (TA150121, OriGene), anti-E2F2 (ab138515, Abcam), and anti-CDKN2C (ab192239, Abcam).
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