MDA-MB-231 cells were pre-treated with 20 μM or 100 μM FC2 for 1 h. Subsequent stimulation with TNF was performed at a concentration of 50 ng/mL. After cells harvesting, cleared supernatants of cells lysates or cytoplasmic/nuclear extracts (obtained with NE-PER™ Nuclear and Cytoplasmic Extraction kit by Thermo Fisher Scientific) were separated by SDS-PAGE on precast 4–12% Bis-Tris NuPAGE gels (Thermo Fisher Scientific) and proteins were transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany) using the XCell II blot module (Thermo Fisher Scientific). Membranes were then saturated for 60 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, phospho-NF-kB-p65 #3033, Myc-Tag #2278; Phospho-MKK3/MKK6 #9236, Phospho-TAK1 #4508 (Cell Signaling) cIAP2, p65 and Vinculin #V9131 (Sigma-Aldrich). After incubation with the proper secondary antibody (anti-rabbit and anti-mouse IgG were purchased from GE Healthcare, UK; all secondary antibodies were diluted 1:2000), proteins were detected by chemiluminescence (EuroClone).
Anti rabbit and anti mouse igg
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Sweden
Anti-rabbit and anti-mouse IgG are secondary antibodies used in immunodetection techniques. They specifically bind to the primary antibodies raised against rabbit or mouse immunoglobulins, respectively, allowing for the detection and visualization of target proteins or molecules in various applications such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Xcell 2 blot module, by Thermo Fisher Scientific (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Myc tag 2278, by Cell Signaling Technology (1 mentions)
Vinculin v9131, by Merck Group (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
2 14c acetic acid, by PerkinElmer (1 mentions)
Acetyl coa carboxylase c83b10, by Cell Signaling Technology (1 mentions)
3h leucine, by PerkinElmer (1 mentions)
35s methionine, by PerkinElmer (1 mentions)
Spark microplate reader, by Tecan (1 mentions)
V9131, by Merck Group (1 mentions)
Ab64575, by Abcam (1 mentions)
Ma1 80216, by Thermo Fisher Scientific (1 mentions)
Sc 544, by Santa Cruz Biotechnology (1 mentions)
5 protocols using anti rabbit and anti mouse igg
1
Investigating NF-κB Signaling in MDA-MB-231 Cells
2
Lipid Metabolism Inhibition Assay
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Doxycycline, palmitic acid, TOFA, c75, MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), and anti-GAPDH antibody were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). FASN (C20G5) and acetyl-CoA carboxylase (C83B10) antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA, USA). Anti-rabbit and anti-mouse IgG, and HRP-linked antibodies were from GE (GE Healthcare Life Science, Pittsburgh, PA, USA). BRG1 antiserum [70 (link)] was used in western blot and ChIP experiments. The BRM antibody (ab15597) used in western blots was purchased from Abcam (Cambridge, MA, USA). D-14C-Glucose, 3H-Leucine, 35S-Methionine and [2–14C] acetic acid were purchased from PerkinElmer (PerkinElmer Life Sciences, Waltham, MA, USA). ADAADi was prepared as described [28 (link)].
3
Western Blot Analysis of Protein Expression
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For Western blot assay, cells were harvested, washed with PBS, boiled for 10 minutes in lysis buffer (125 mmol/L Tris HCl pH 6.8, 5% of SDS) and supplemented with protease inhibitors (11873580001, cOmplete, Roche). Samples were then sonicated for 20 seconds, followed by centrifugation for 15 minutes at 13,000 rpm and quantified with micro BCA Assay kit reagent (23235, Thermo Fisher Scientific) using the Spark microplate reader (TECAN). Proteins were separated by SDS-PAGE on precast 4% to 12% Bis-Tris NuPAGE gels (NP0321PK2, Thermo Fisher Scientific) and blotted to Immobilon PVDF membranes (IPVH00010, Merck Millipore) using the XCell II blot module (EI9051, Thermo Fisher Scientific). Membranes were then saturated for 1 hour in Tris-buffered saline containing 5% BSA and incubated overnight with the antihuman primary antibodies recognizing: JMJD6 (ab64575, Abcam and P1495, Sigma-Aldrich), Actin-β (A1978, Sigma-Aldrich), ERα (sc-544, G20, Santa Cruz Biotechnology and MA1–80216, Invitrogen), ANXA1 (ab214486, Abcam) and Vinculin (V9131, Sigma-Aldrich). After hybridization with the appropriate secondary antibody (anti-rabbit and anti-mouse IgG, GE HealthCare), proteins were detected by chemiluminescence (EuroClone).
4
Western Blot Analysis of APP Glycosylation
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The samples were subjected to SDS-PAGE (10% or 5%–20% gradient gel) and transferred to nitrocellulose membranes. For Western blot analyses, following incubation with 5% non-fat dried milk in TBS-containing 0.1% Tween-20, the membranes were incubated with anti-APP 22C11 (1:1000 dilution), anti-APP(C) (1:200–1000 dilution), anti-O-GlcNAc RL2 (1:500 dilution), or anti-actin (1:500 dilution) antibodies. Appropriate horseradish peroxidase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) or anti-mouse and anti-rabbit IgG (GE Healthcare) antibodies were used as the secondary antibodies (1:1000 dilution). For the lectin pull-down experiment, the lysates (50 μg of protein) were incubated with 20 μl of Arachis hypogaea (PNA)-coupled agarose (J-oil Mills), and subjected to Western blot analysis with anti-APP(C). Signals were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) using ImageQuant LAS-4000mini (GE Healthcare). The intensity of the resultant protein bands was quantified using ImageQuant TL software (GE Healthcare).
5
Western Blot and ELISA for HIF-1α Quantification
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Protein lysates were extracted using RIPA buffer (Thermo Fisher, Waltham, MA) with protease inhibitor cocktail (Roche, Basel, Switzerland). Thirty μg of protein was boiled with 5x Laemli buffer (0.5 M Tris-HCl, 10% SDS, 30% glycerol, 0.25% Bromphenol blue, 20% β-mercaptoethanol) for 10 mins. Protein samples were separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose membranes (GE Healthcare, Danderyd, Sweden). After milk blocking, blots were incubated over night at 4°C with the following primary antibodies: PPZ0506 [17 (link)] for ERβ2 (R&D systems, Abingdon, UK), PHD3 (Novus biological, Littleton, CO), HIF-1α (Pharmingen, San Diego, CA) and β-actin (Sigma Aldrich, Dorset, UK). Secondary antibodies were anti-mouse and anti-rabbit IgG (GE Healthcare, Danderyd, Sweden).
For ELISA analysis, thirty μg of protein was used. HIF-1α ELISA was performed using Human/Mouse Total HIF-1 alpha DuoSet IC ELISA kit (R&D systems, Abingdon, UK) according to manufacturer's instructions.
For ELISA analysis, thirty μg of protein was used. HIF-1α ELISA was performed using Human/Mouse Total HIF-1 alpha DuoSet IC ELISA kit (R&D systems, Abingdon, UK) according to manufacturer's instructions.
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