Cells were lysed with 200 μl of Lysis buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF). 18 μl samples were then loaded onto gels alongside 8 μl of protein ladder. The proteins were then transferred to nitrocellulose membranes before incubation in blocking buffer (5% (w/v) skimmed milk powder in TBS-T) for 1 hour at room temperature. The blots were then washed in TBS-T and incubated overnight with anti-NFATc2 (NFAT1) (BD Transduction Laboratories) or anti-phospho-p65 (S536) (Cell Signaling Technologies) primary antibodies (BD Transduction Laboratories) at 1:5000 and 1:1000 dilution respectively, in 5% (w/v) BSA in TBS-T. Blots were washed in TBS-T and then incubated for a further hour with a 1:2000 dilution of HRP-conjugated anti-IgG (Cell Signaling Technology). After a final TBS-T wash to remove any unbound antibody, a 1:1 ratio mix of ECL Advance Western Blotting Detection kit reagents was then applied to the membrane and left for 1 minute, before visualizing HRP-conjugated proteins using a Bio-Rad Gel Doc XRS+ System. Membranes were then re-stained with an anti-α-Tubulin primary antibody (Cell Signaling Technologies) and the detection process was repeated in order to produce a loading control.
Anti phospho p65 s536
Manufactured by Cell Signaling Technology
Sourced in China, Japan
Anti-phospho-p65 (S536) is a primary antibody that detects the phosphorylated form of the p65 subunit of the NF-kB transcription factor. It recognizes p65 when phosphorylated at serine 536.
Automatically generated - may contain errors
Lab products found in correlation
Anti nfatc2 nfat1, by BD (2 mentions)
Gel doc xrs system, by Bio-Rad (2 mentions)
Anti α tubulin primary antibody, by Cell Signaling Technology (1 mentions)
Anti total p65, by Abcam (1 mentions)
Anti inos, by GeneTex (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342 trihydrochloride trihydrate, by Thermo Fisher Scientific (1 mentions)
Anti phospho iκbα, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti ck2α, by Cell Signaling Technology (1 mentions)
Anti phospho stat3, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Dc assay, by Bio-Rad (1 mentions)
A3854, by Merck Group (1 mentions)
Sab3500023, by Merck Group (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti phospho p65 s536
1
Western Blot Analysis of NFAT and NF-κB Signaling
2
Intracellular Protein Detection by Western Blotting
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Western blotting was performed to detect the intracellular proteins. The proteins of different THP-1 treatment groups were harvested using the RIPA (radioimmunoprecipitation assay) lysis buffer, and the concentration was determined by a BCA Protein Assay Kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to distinguish the proteins by their KD size, and the quantity was determined by the gray level of banding. The primary antibodies were anti-phospho-P65 (S536) (Cat# 3033, Cell Signaling Technology, Shanghai, China), anti-total-p65 (Cat# ab7970, Abcam, Cambridge, UK), and anti-iNOS (Cat# 821505270, GeneTex, Beijing, China), and anti-GAPDH was used as a control (Cat# 60004-1-lg, ProteinTech). The secondary antibodies were purchased from Jackson ImmunoResearch Inc. (Cat# 111-035-003 for anti-Rabbit, Cat# 115-035-003 for anti-Mouse, West Grove, PA, USA).
3
Antibody Validation for Molecular Analyses
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The following antibodies (Abs) were used for western blotting, immunoprecipitation, qPCR, ELISA, confocal microscopy, immunohistochemistry, flow cytometry, or joint injection: anti-TMEM147 (sc-138814, Santa Cruz Biotechnology), anti-TMEM147 (generated by immunizing rabbit with peptide containing amino acids 121-136 VGARGIEFDWKYIQMSC, Sigma-Aldrich), anti-NF-κB p65 (sc-372, Santa Cruz Biotechnology), anti-phospho-p65 S536 (#3033, Cell Signaling Technology, Tokyo, Japan), anti-phospho NF-κB p65 S529 (ab47395, Abcam, Tokyo, Japan), anti-phospho NF-κB p65 S276 (SAB4504488, Sigma-Aldrich), anti-phospho-stat3 (#9145, Cell Signaling Technology), anti-IκBα (#4814, Cell Signaling Technology), anti-phospho-IκBα (#9246, Cell Signaling Technology), anti-BCR (#3902, Cell Signaling Technology), anti-phospho-BCR Y177 (#3901, Cell Signaling Technology), anti-CK2α (#2656, Cell Signaling Technology), anti-phospho-CK2α (SAB4300628, Sigma-Aldrich), anti-α-tubulin (T5168, Sigma-Aldrich), anti-FLAG M2 (F1804, Sigma-Aldrich), rabbit IgG (#3900, Cell Signaling Technology), anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology), anti-mouse IgG (sc-2314, Santa Cruz Biotechnology), anti-goat IgG (sc-3851, Santa Cruz Biotechnology), Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A11055, Life technologies, Tokyo, Japan) and Hoechst 33342 trihydrochloride trihydrate (H3570, Life technologies).
4
Western Blot Analysis of NFAT and NF-kB Signaling
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Cells were lysed with 200 μl of lysis buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF). Samples (18 μl) were then loaded onto gels alongside 8 μl of protein ladder. The proteins were then transferred to nitrocellulose membranes before incubation in blocking buffer (5% [w/v] skimmed milk powder in TBST) for 1 h at room temperature. The blots were then washed in TBST and incubated overnight with anti-NFATc2 (NFAT1) (BD Transduction Laboratories) or anti–phospho-p65 (S536) (Cell Signaling Technology) primary Abs (BD Transduction Laboratories) at 1:5000 and 1:1000 dilution, respectively, in 5% (w/v) BSA in TBST. Blots were washed in TBST and then incubated for a further hour with a 1:2000 dilution of HRP-conjugated anti-IgG (Cell Signaling Technology). After a final TBST wash to remove any unbound Ab, a 1:1 ratio mix of ECL Advance Western blotting detection kit reagents was then applied to the membrane and left for 1 min, before visualizing HRP-conjugated proteins using a Bio-Rad Gel Doc XRS+ system. Membranes were then restained with an anti–α-tubulin primary Ab (Cell Signaling Technology), and the detection process was repeated to produce a loading control.
5
Apabetalone modulates NF-κB signaling in HUVECs
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HUVECs were treated with apabetalone (5 or 20 μM) or DMSO and TNFα (10 ng/ml) for 2 h. Nuclear and cytoplasmic lysates were prepared using NE-PER kit (ThermoScientific, 78833) with freshly added protease inhibitor cocktail (BioShop or Sigma-Aldrich), phosSTOP phosphatase inhibitor (Roche 04906837001), and 0.5uM TSA (HDAC inhibitor). Protein concentration was determined with BioRad DC assay and lysate was added to NuPAGE LDS sample buffer (Novex/Invitrogen/Life Technologies NP0007) and 20 ug of total protein was loaded onto a NuPAGE 4-12% Bis-Tris gel (Novex/Invitrogen/Life Technologies NP0321BOX). For immunoblotting, the following primary antibodies were used: anti-p65 (Abcam, ab16502), anti-phospho p65 S536 (Cell Signaling, 3033), anti-BRD2 (Bethyl, A302-583A), anti-alpha tubulin (Sigma, SAB3500023), and anti-β-actin conjugated to peroxidase (Sigma, A3854). Secondary antibodies used were goat anti-rabbit IgG H&L chain specific peroxidase (Calbiochem, 401353) and rabbit anti-chicken IgY H&L chain specific peroxidase (Abcam, ab6753). Immunoreactive proteins were visualized by the chemiluminescent reagent ECLTM prime (GE Healthcare, RPN2232).
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