IL-4– and/or mechanical loading-treated human chondrocytes were lysed for western blot analysis. Approximately 20 μg of total protein extracted from cell lysates was separated by electrophoresis (SDS-PAGE) in a 10% polyacrylamide gel, and the separated proteins were transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA). Membranes were incubated with primary antibodies specific for JAK3, STAT6, phosphorylated JAK3, and STAT6 (all from Cell Signaling), and for MMP13 (Abcam) and CITED2 (Santa Cruz), followed by incubation with secondary antibodies conjugated to horseradish peroxidase, and detected by an ECL western blotting analysis system (GE Life Sciences, Piscataway, NJ). A rabbit antibody specific for human β-actin (Sigma-Aldrich, St. Louis, MO) was used as a loading control.
Cited2
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom
CITED2 is a protein that functions as a transcriptional coactivator, enhancing the activity of various transcription factors. It plays a role in cellular processes such as development, hypoxia response, and regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting analysis system, by GE Healthcare (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Snail, by Santa Cruz Biotechnology (1 mentions)
Twist, by Santa Cruz Biotechnology (1 mentions)
Prmt5, by Santa Cruz Biotechnology (1 mentions)
Anti erg, by Abcam (1 mentions)
N cad, by Santa Cruz Biotechnology (1 mentions)
Anti myc, by Merck Group (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti flag, by Merck Group (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Merck Group (1 mentions)
4 protocols using cited2
1
Chondrocyte Signaling Pathway Analysis
2
Antibodies and Reagents for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against CITED2, PRMT5, β-tubulin, WDR77, RioK1, p300, β-CTN, Vimentin, TWIST, Snail, N-Cad, and ZEB1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-NCL, anti-dimethyl-arginine and anti-acetyl-lysine from Upstate Biotechnology (Lake Placid, NY); anti-p-AKT, anti-AKT and anti-E-cadherin from Cell Signaling (Danvers, MA); anti-FLAG, anti-MYC, and anti-HA from Sigma-Aldrich (St. Louis, MO); and anti-ERG and anti-α-SMA from Abcam (Cambridge, MA). MK2206 was purchased from Selleckchem. Human recombinant proteins of CITED2, PRMT5, P300, and NCL were purchased from Origene. Fetal bovine serum (FBS), dithiothreitol, G418 disulfate salts (G418), EPZ015666, Leptomycin B, Bay-11-7082, LY294002, Cycloheximide, Wortmannin, and others were obtained from Sigma-Aldrich. Sources and dilution factors of antibodies used are summarized in Supplementary Table 1 .
3
Immunoblotting Analysis of Cellular Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting were performed as previously described (3 (link),33 (link)). The following primary antibodies were used at dilutions of 1:1,000 to 1:2,000 in 5% nonfat milk: CITED2 (Santa Cruz Biotechnology), CHOP, phospho-(p-)PERK, (p-)IRE1α, (p-)eIF2α (eukaryotic translation initiation factor 2A), and cleaved caspase 3 (Cell Signaling Technology). Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Scientific). All experiments were repeated in triplicate. Antibody information is listed in Supplementary Table 2 .
4
Protein Analysis of Cartilage and CEP Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein from chondrocytes or CEP tissue was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China). Protein quantification was performed with the BCA Protein Quantitation Kit (Beyotime, China). Next, the protein was separated on an SDS-polyacrylamide gel before transferring into a PVDF membrane (Millipore, United States). The membrane was then blocked with skim milk, incubated overnight at 4°C with primary antibody, washed 3×, and incubated at room temperature for 2 h with secondary antibody. The antibodies used in this study were collagen type II (1:3,000; Abcam, United Kingdom), aggrecan (1:1,000; Abcam, United Kingdom), MMP-13 (1:3,000; Abcam, United Kingdom), CITED2 (1:500; Santa Cruz Biotechnology), GAPDH (1: 5,000; Abcam, United Kingdom), and goat anti-rabbit secondary antibody (1:5,000; Abcam, United Kingdom). The protein bands were quantified using Image J software and GADPH was used as a loading control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!