Total protein was extracted from cultured cells and quantified using Trizol reagent (Invitrogen) following the protocols. Equal amounts of protein from different cells were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% non-fat milk and incubated with antibodies against MHC-ІІ (ab55152, Abcam), IL-1β (#12242, CST), CD206 (ab64693, Abcam), Arg-1 (#9819, CST), GAPDH (ab8245, Abcam), CEBPA (ab40764, Abcam), and β-actin (ab8245, Abcam) at the concentration of 1:1000 at 4°C overnight. The blots were then incubated with horseradish peroxidase–conjugated secondary antibodies in blocking buffer at room temperature for 2 h. The signal was detected using ECL system (Amersham Pharmacia) and quantified by scanning densitometry and computer-assisted image analyzer.
Ab40764
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab40764 is an antibody product offered by Abcam. It is a recombinant monoclonal antibody targeting a specific antigen. The core function of this product is to facilitate detection and analysis of the target antigen in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Sc 61, by Abcam (2 mentions)
Ab32358, by Thermo Fisher Scientific (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab92501, by Abcam (2 mentions)
Ab59256, by Abcam (2 mentions)
Ab32391, by Abcam (2 mentions)
Ab76956, by Abcam (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ecl system, by Cytiva (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab55152, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Phenol chloroform isoamyl alcohol, by Merck Group (1 mentions)
Bioruptor sonicator, by Diagenode (1 mentions)
4000r imaging station, by Kodak (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab9482, by Abcam (1 mentions)
Ab28146, by Abcam (1 mentions)
16 protocols using ab40764
1
Protein Extraction and Western Blot Analysis
2
ChIP-qPCR on Stamp2 Promoter
3
Protein Extraction and Western Blot Analysis
4
Western Blot Analysis of Protein Signaling
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Total protein lysates were prepared as previously described.3 , 29 , 30 Proteins were separated by SDS‐PAGE and transferred to the nitrocellulose membranes. Then the membranes were incubated with antibodies against CYP2E1 (Abcam; ab28146, 1:5000), phosphor‐p44/42 MAPK (Erk1/2) (Thr201/Tyr204) (Cell Signaling; 4370, 1:2000), p44/42 MAPK (Erk1/2) (Cell Signaling; 9102, 1:1000), phosphor‐PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cell Signaling; 4228, 1:1000), PI3 Kinase p85 (Cell Signaling; 11889, 1:1000), phosphor‐Akt (Ser473) (Cell Signaling; 4060, 1:2000), Akt (Cell Signaling; 8596, 1:1000), Myc (Abcam; ab17355, 1:500), CEBP Alpha (Abcam, ab40764), CEBP Beta (Abcam, ab32358), or C/EBP zdta (ThermoFisher, PA5‐37925). Antibody binding was detected with horseradish peroxidase–conjugated immunoglobulin G using a chemiluminescence detection system after incubation with the appropriate secondary antibodies. GAPDH was used as normalization (Abcam; ab9482, 1:5000). Bands were quantitatively analyzed with ImageJ software.
5
Cell Lysis and Protein Extraction
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Cells were harvested in a 24-well-plate format (in triplicates) for a pool of 3 wells per condition for total protein extraction. Prior to cell lysis, the wells are washed twice with cold PBS and transferred into pre-chilled tubes with the use of a cell scraper. The cells were pelleted gently at 3,500 rpm for 5 min at 4°C before addition of RIPA lysis buffer containing 50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP40, and protease inhibitor cocktail (Sigma). Cells were incubated for 10 min on ice, followed by vortexing for 2 min to allow complete cell lysis. Cell debris was then removed by centrifugation at 10,000 rpm for 10 min at 4°C. The protein supernatant was then transferred into a clean pre-chilled tube. Protein amount per sample was quantified using the RC-DC Bradford assay kit following the manufacturer’s protocol (Bio-Rad), and 50 μg of total protein was loaded for SDS-PAGE. The acrylamide gels were then transferred onto polyvinylidene fluoride (PVDF) membranes for western blotting using the following antibodies: C/EBP-α, ab40764 (Abcam); β-tubulin, ab6046 (Abcam); and anti-rabbit-HRP, 926-8011 (LI-COR).
6
Comprehensive Protein Expression Analysis
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Calnexin, VEGFA, CD31, HIF-1 alpha, (AB0037, AB0063, AB0092, AB0112, Sicgen, Portugal), β-actin, α-tubulin (A5316, T6199, Sigma, USA), PPARγ, AKT, p-AKT(Ser473), VEGFR2, PI3K (#2443, #9272, #4058, #2479, #4249, Cell Signaling, USA), ANG-2, F4/80, Perilipin-A, GLUT4, p-IR(Y1361), GLO-I, C/EBPalpha, PGC1alpha, ANGPTL4, AT1, HIF-2 alpha (Ab8452, Ab74383, Ab3526, Ab65267, Ab60946, Ab96032, Ab40764, Ab191838, Ab2920, Ab9391, Ab8365, Abcam, UK), CD11c, CD206 (bs-2058R, bs-2664R Bioss, USA) TIE-2, IRβ (sc-324, sc-57342, SantaCruz Biotechnology, USA), CEL (KH025, TransGenic Inc, Japan).
7
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using RIPA lysis buffer supplemented with protease inhibitor (Solarbio, Beijing, China). Then, the extracts were separated by 10–12% SDS/PAGE and transferred to PVDF membranes. The membranes were blocked with 10% non‐fat milk powder in TBS for 2 h at room temperature and incubated with anti‐GRK6 (1 : 500, sc‐566; Santa Cruz Biotechnology, Dallas, TX, USA), anti‐C/EBPα (1 : 500, ab40764; Abcam), anti‐E‐cadherin (1 : 1000, ab1416; Abcam), anti‐vimentin (ab92547; Abcam), and anti‐β‐actin (1 : 5000, ab8226; Abcam) primary antibodies at 4 °C overnight. After three washes with TBST, the membranes were incubated with horseradish peroxidase‐conjugated goat anti‐mouse IgG (1 : 5000, sc‐2005; Santa Cruz Biotechnology) and horseradish peroxidase‐conjugated goat anti‐rabbit IgG (1 : 5000, sc‐20045; Santa Cruz Biotechnology). The signal intensity of the bands was calculated on grayscale images using imagej .
8
Quantifying Adipocyte Markers and Apoptosis
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3T3-L1 cells were lysed with RIPA buffer (Product number: R0010; Solarbio), and the protein concentration was quantified with a BCA protein assay kit (Product number: PC0020; Solarbio). For western blot analysis, equal amounts of protein were loaded on a 10% sodium dodecyl sulfate polyacrylamide gel electro-phoresis gel and then electrotransferred to polyvinylidene fluoride membranes (Product number: 1620177; Bio-Rad, CA, USA). The membranes were blocked with 5% nonfat milk for 90 min and then treated with antibodies against PPARγ (1:1,000, ab178866; Abcam, CA, USA), C/EBPα (1:800, ab40764; Abcam), BCL-2 (1:1,500, ab196495), BAX (1:3,000, ab32503), and β-actin (1:2,000, ab8226; Abcam) overnight at 4°C. An anti-rabbit secondary antibody (1:8,000, ab288151; Abcam) was used to treat the membranes, and an ECL kit (Pierce, IL, USA) was applied to visualize the membranes.
9
Protein Expression Analysis in DRGs
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The DRGs were homogenated with tissue lysate plus protease inhibitors according to the manufacturer’s instruction (Promega, Madison, WI, USA), and then centrifuged and collected the supernatant and determined concentration of the protein (Bio-Rad, Hercules, CA, USA). By SDS-PAGE gel, the protein samples (30 μg) were separated and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% milk and then incubated with rabbit anti-NGF antibody at 4 °C overnight (1:1000, ab6199, Abcam), rabbit anti-C/EBPα antibody (1:1000, ab40764, Abcam), rabbit anti-DNMT3a antibody (1:800, ab4897, Abcam), rabbit anti- DNMT3b antibody (1:1000, ab2851, Abcam), or mouse anti-β-actin antibody (1:5000, ab8227, Abcam). After that, the membrane was also incubated with secondary antibodies (1:10000, goat anti-mouse IgG (H + L) or donkey anti-rabbit IgG (H + L) labeled with IRDye 800CW). The blot intensity was analyzed using Odyssey (Li-COR, USA) for the gray scale value statistics. The protein expression was normalized to β-actin.
10
PU.1 Interacting Protein Complex Analysis
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Immunoprecipitation assays were performed using an immunoprecipitation kit (Abcam, ab206996) and following the manufacturer's instructions. Briefly, the cells were lysed in RIPA buffer, and anti‐human PU.1 antibody (1:100, Thermofisher Scientific, PA5‐17505) was added into the lysate before overnight incubation at 4 °C. Protein G‐conjugated beads were added to pull down the specific protein complex interacting with PU.1. Western blot analysis was performed on the eluted complex using anti‐human C/EBPα/β (1:1000, Abcam, ab40764, and ab32358) and anti‐human SMARCB1 (1:1000, Thermofisher Scientific, PA5‐40834).
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