Western blot analysis was conducted using the procedure previously described 49 (link). We used antibodies against, β-catenin, Lrp5, Runx2, Snail, TGFβ, MSN, PDL1, CD44 (Cell Signaling, Danvers, MA, USA), MMP9, NFATc1, cathepsin K, OPN, alkaline phosphatase, FN1 (Santa Cruz Biotechnology), CD95, RANKL, OPG (Invitrogen, Carlsbad, CA, USA), Hsp90ab1, Osteocalcin (Abcam, Cambridge, UK), KDM3A (Proteintech, Rosemont, IL, USA), and β-actin (Sigma). The levels of Hsp90ab1 and MSN in Lrp5 CM were determined using ELISA kits (MBS7700502 and MBS2124159; MyBioSource). The level of PDL-1 in EO771 cells, which were treated with 0.05, 0.1, 0.5, and 1 μg/mL of TGFβ and Lrp5 CM, was determined using a cell ELISA kit (PI62200; Invitrogen).
Kdm3a
Manufactured by Proteintech
Sourced in United Kingdom, United States
KDM3A is a histone demethylase enzyme that catalyzes the removal of methyl groups from histone H3 at lysine 9 (H3K9). This enzymatic activity regulates gene expression and chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
Hif 1α, by BD (2 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Osteocalcin, by Abcam (1 mentions)
Tgf β, by Cell Signaling Technology (1 mentions)
Pd l1, by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Hsp90ab1, by Abcam (1 mentions)
Nfatc1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Ab23366, by Abcam (1 mentions)
Ab76118, by Abcam (1 mentions)
γ tubulin gtu 88, by Merck Group (1 mentions)
Nb100 77282, by Novus Biologicals (1 mentions)
β actin, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Cleaved parp, by Abcam (1 mentions)
Cleaved casp3, by Cell Signaling Technology (1 mentions)
0.45 m polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti rabbit immunoglobulin g igg 7074, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using kdm3a
1
Comprehensive Protein Analysis Using Western Blot
2
Antibody Characterization for Protein Methylation
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The antibodies used along this study are to KDM3A (12835; Proteintech; and NB100-77282; Novus Biologicals, Littleton, CO); KDM3B (IHC 00189; Bethyl Laboratories, Montgomery, TX); Cct4 (ARP34271; Aviva); anti–mono- and dimethylated lysines (ab23366 and ab76118; Abcam, Cambridge, MA); anti-GFP (sc-8334), monoclonal to GAPDH (5019A-2; Imgenex, San Diego, CA), β-gal (A-11132; Molecular Probes), GST (C83271; LSBio); HP1a (clone 15.1952; Upstate); γ-tubulin (GTU-88; Sigma-Aldrich, St. Louis, MO), β-actin (ab8229; Abcam), Hsp90ab1 (MAB32861; R&D Systems), Hsp90aa1 (10713715; Pierce, Rockford, IL), Actbl2 (ab134977; Abcam). Secondary antibodies: anti-mouse and anti-rabbit F(ab′)2 immunoglobulin G Alexa Fluor 488 and 584 (Molecular Probes) for immunofluorescence or horseradish-conjugated peroxidase for immunoblots.
3
Quantifying Protein Expression by RIPA Extraction
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The extracted total protein in cells and tissues by radio-immunoprecipitation assay (RIPA) buffer was quantified by bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Subsequently, total protein was separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a 0.45-µM polyvinylidene fluoride membrane (MilliPore, MA, USA). The protein membrane incubated with the specific antibodies were developed by enhanced chemiluminescence and exposed by Image Quant LAS 4000C (GE Company, USA). Primary antibodies included KDM3A (1:200, 12,835–1-AP, Proteintech), HIF-1α (1:1000, 610,958, BD Biosciences), Cleaved-PARP (ab32064, Abcam, MA, USA), Cleaved-CASP3 (9661, Cell Signaling Technology) and β-actin (1:1000, sc-47778, Santa Cruz Biotechnology) while secondary antibody included anti-rabbit immunoglobulin G (IgG; 7074, 1:2000, Cell Signaling Technology). β-actin was the internal control [24 (link)].
4
Immunoprecipitation and Western Blotting
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Cells were lysed with IP lysis buffer (500 μL) on ice and centrifuged at 13,500 r/min. The supernatant was reacted with the antibody overnight, added with protein A + G agarose (P2055, Beyotime, China) and centrifuged at 13,500 r/min. After washing 3 times with cold lysis buffer, the immunoprecipitate was combined with sodium dodecyl sulfate, heated to 98 °C and treated with sodium dodecyl sulphate polyacrylamide gel electrophoresis. After that, the protein was transferred to a 0.22-μm polyvinylidene fluoride membrane, immersed in 10% skimmed milk and combined with the primary antibodies KDM3A (1:200, 12,835–1-AP, Proteintech) and HIF-1α (1:1000, 610,958, BD Biosciences). Then, the membrane was washed 3 times with Tris-buffered saline with Tween-20 and cultured with IgG antibody (7074, 1:2000, Cell Signaling Technology). The pellet was rinsed with RIPA buffer and then subjected to Western blot analysis. Normal IgG was used as a NC [26 (link)].
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