Western blotting procedures were carried out as previously described (15 ). Briefly, cells were collected in RIPA lysis buffer (150mM NaCl, 1% NP40, 0.5% DOC, 50mM Tris-HCl at pH 8, 0.1% SDS, 10% glycerol, 5mM EDTA, 20mM NaF and 1mM Na3VO4, 1 μg/ml each of pepstatin, leupeptin, and aprotinin, 200 μg/ml phenyl-methylsulfonyl-fluoride). Lysates were cleared by centrifugation at 14,000 x g for 20 minutes at 4 °C and analyzed by SDS-PAGE and autoradiography. Lysates from mammospheres were collected 7 days after culture in mammosphere media. Corresponding adherent controls were put on mammosphere media 24 hrs after seeding and collected the next day. Antibodies were purchased from the following companies; Anti-OGT (Santa Cruz, Cell-Signaling), Anti-O-GlcNAc (Santa Cruz, Sigma), Anti-c-Myc (Novus), Anti-Actin (Santa Cruz), Anti-CD44, Anti-Fibronectin, Anti-Vimentin, Anti-NANOG (Cell-Signaling), Anti-K8/18 (Thermo-Fisher), Anti-K14 (Thermo-Fisher), Anti-GAPDH (Genscript), Anti-KLF8 (Sigma). Densitometry was performed using Image J Software (National Institutes of Health, Bethesda, MA).
Anti c myc
Manufactured by Novus Biologicals
Sourced in United States
Anti-c-MYC is a laboratory reagent used to detect and quantify the c-MYC protein, a transcription factor involved in cell growth and proliferation. It can be used in various techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of c-MYC in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti o glcnac, by Merck Group (3 mentions)
Anti hif 1α, by Novus Biologicals (3 mentions)
Anti nanog, by Cell Signaling Technology (2 mentions)
Anti klf8, by Merck Group (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Methyl pyruvate, by Merck Group (2 mentions)
Anti ubiquitin, by Cell Signaling Technology (2 mentions)
Anti s6 ribosomal protein, by Cell Signaling Technology (2 mentions)
Anti acc, by Cell Signaling Technology (2 mentions)
Anti acly, by Abcam (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti 4e bp1, by Cell Signaling Technology (2 mentions)
Anti ogt, by Merck Group (2 mentions)
Anti fas, by Cell Signaling Technology (2 mentions)
Anti psrebp1, by Merck Group (2 mentions)
Anti praptor, by Cell Signaling Technology (2 mentions)
Anti p ampk, by Merck Group (2 mentions)
Anti ps6 ribosomal protein, by Cell Signaling Technology (2 mentions)
Anti srebp1, by Abcam (2 mentions)
Dorsomorphin 2hcl, by Selleck Chemicals (2 mentions)
7 protocols using anti c myc
1
Western Blotting of Mammosphere Proteins
2
Protein Expression Analysis in Stem Cells
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Cells were collected and washed three-times with ice-cold PBS to remove residue media. Then cell pellets were resuspended in cold RIPA lysis buffer (150mM NaCl, 1% NP40, 0.5% DOC, 50mM Tris-HCl at pH 8, 0.1% SDS, 10% glycerol, 5mM EDTA, 20mM NaF and 1mM Na3VO4), supplemented with protease inhibitors. Cell debris was removed by centrifugation at 15000 rpm for 20 minutes at 4°C using benchtop centrifuge. Protein concentration was determined flowing Bradford assay. 50 µg of protein from each sample was separated by SDS-PAGE and transferred to PVDF membrane. Target proteins were detected using indicated specific antibodies: Anti-OGT (Cell-Signaling, cat #20438), Anti-O-GlcNAc (Sigma, cat #MABS1254), Anti-c-MYC (Novus, cat #NB600-335), Anti-Actin (Santa Cruz, cat #sc-47778), Anti-NANOG (Cell-Signaling, cat #3580), Anti-SOX2 (Cell-Signaling, cat #3579), Anti-OCT4 (Cell-Signaling, cat #2750), Anti-KLF8 (Sigma, cat #AV32859).
3
Immunostaining for HIF-1α, c-Myc, YY1, MDR1
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Immunostaining was performed as previously describe [28 (link)]. Anti-HIF-1α, anti-c-Myc, anti-YY1 or anti-MDR1 (Novus Biological, Litletown, CO, USA).
4
Regulation of SREBP1 Signaling
5
Regulation of SREBP1 Signaling
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MG132, Nile Red, Methyl-pyruvate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C and Dorsomorphin (2HCL) from Selleck-Chemicals (Houston, TX, USA). Ac-5sGlcNAc was provided by D. Vocadlo (Simon Fraser University). Antibodies used were Anti-OGT, Anti-O-GlcNAc, Anti-FLAG from Sigma-Aldrich; Anti-pSREBP1-(S372), Anti-pAMPK-(T172), Anti-pRaptor-(S792), Anti-pS6 Ribosomal Protein-(S240/244), Anti-p4EBP1-(T70), Anti-AMPK, Anti-Raptor, Anti-S6 Ribosomal Protein, Anti-4EBP1, Anti-FAS, Anti-ACC, Anti-Ubiquitin, Anti-Cleaved Caspase 3, Anti-Cleaved PARP from Cell Signaling (Danvers, MA, USA); Anti-Actin, Anti-Bcl2 from Santa Cruz Biotechnology; Anti-SREBP1, Anti-HIF1α, Anti-c-MYC from Novus Biologicals; Anti-SREBP1, Anti-ACLY, Anti-Glut1 from Abcam; Anti-FBW7 from Bethyl Labs. pLKO.FLAG-SREBP1 (Addgene-32017 from D. Sabatini).
6
Western Blot Analysis of Protein Markers
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Proteins were extracted with lysis buffer (Tri-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% NaDOC, 0.1% SDS, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride, 1 μg ml−1 pepstatin, 1 μg ml−1 aprotinine and 10 μg ml−1 leupeptine) for 20 min on ice, and protein amounts were quantified using Micro BCA Protein Assay Reagent (23235; Pierce Chemical Co.). Whole-cell lysates (40 μg per lane) were resolved on SDS–polyacrylamide gels, electrotransferred onto polyvinylidene difluoride membranes and examined by immunoblot analysis. The primary antibodies used were anti-PAX5 (610862; BD PharMingen), anti-BCL6 (#4242; Cell Signaling), anti-c-MYC (NB600-302C; Novus Biologicals), anti-IRF4 (sc-6059; Santa Cruz), anti-BLIMP1 (NB600-235; Novus Biologicals), anti-XBP1 (sc-7160; Santa Cruz), anti-HO-1 (NBP1-97507; Novus Biologicals), anti-AID (provided by Dr Alt) and anti-GAPDH (#2118; Cell Signaling). Full-sized scans of all western blots shown in Figs 3 and 6 and Supplementary Figs 11 and 12 are provided in Supplementary Fig. 14 . Quantification of intensity was performed using an Image J (http://imagej.nih.gov/ij/ ).
7
Protein Extraction and Western Blot Analysis
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Cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Beyotime, China) to extract total proteins. After quantification, 25 μg of protein was loaded and separated with 4–12% Bis-Tris SurePAGE gels (GenScript, China) and then transferred onto PVDF membranes (Merck Millipore, Germany). After blocking with 5% BSA for 1.5 h, the membranes were incubated overnight at 4°C with anti-Sox9 (1:1000, Abcam, UK), anti-Sox2 (1:1000, Proteintech, USA), anti-Klf4 (1:1000, SAB, USA), anti-cMyc (1:1000, Novus, USA), anti-TLR4 (1:1000, Proteintech, USA), anti-YAP1 (1:1000, CST, USA), anti-CTGF (1:1000, Abcam, UK), and anti-β-actin (1:4000, Bioworld, USA) antibodies. After incubation with peroxidase-conjugated secondary antibodies (1:5000, Bioworld, USA) for 1.5 h at room temperature, the membranes were visualized using enhanced chemiluminescence detection reagents (GE Healthcare, USA).
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