Cell extracts were prepared by lysing cells cultured in 6-well plates with RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% NaDOAc, 0.1% SDS, and 1.0%NP-40) plus phosphatase and protease inhibitor cocktails (Xpert P3200-001 and P3100-001, respectively, GenDEPOT). Protein concentrations were determined by the Bio-Rad method (DC Protein Assay), and equal amounts of proteins were subjected to SDS-PAGE gels (8–15%). Proteins were transferred onto PVDF membranes and incubated with primary antibody overnight at 4 °C, followed by a 1 h incubation with secondary goat anti-mouse or goat anti-rabbit Alexa-Fluor 680 (Molecular Probes), or donkey anti-goat 780 (LI-COR Biosciences). Primary antibodies include NLRP3 antibody (Adipogen), phospho-p65 antibody, IκB antibody, total p65 antibody and phospho-p38 antibody (Cell Signaling Technology), total p38 antibody and β-actin antibody (Santa Cruz Biotechnology). The results were visualized using Li-Cor Odyssey Infrared Imaging System (LI-COR Biosciences).
Phospho p65 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Phospho-p65 antibody is a laboratory reagent used to detect the phosphorylated form of the p65 subunit of the NF-κB transcription factor. This antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the activation and regulation of the NF-κB signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse or goat anti rabbit alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat 780, by LI COR (1 mentions)
Iκb antibody, by Cell Signaling Technology (1 mentions)
Total p38 antibody, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Nlrp3 antibody, by Adipogen (1 mentions)
Chemiluminescence system, by Cytiva (1 mentions)
Fv1000, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Triton x 100, by Sangon (1 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
Poly d lysine solution, by Sangon (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Fujifilm (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Erk1 2 antibody, by Cell Signaling Technology (1 mentions)
P38 mapk antibody, by Cell Signaling Technology (1 mentions)
5 protocols using phospho p65 antibody
1
Western Blot Analysis of Cellular Proteins
2
Immunoblot Analysis of Cell Signaling
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All the protein samples were subjected to electrophoresis by SDS-PAGE method and then transferred to PVDF membrane for further immunoblot. The primary antibodies used in this study include: RIP3 antibody (Cat. # ab56164; Abcam, Cambridge, UK; 1:1000), phospho-RIP3 antibody (Cat. # ab56164; Abcam; 1:1000), CCR5 (Cat. # ab65850; Abcam; 1:1000), Tublin (Cat. # ab65850; Abcam; 1:1000), phospho-p65 antibody (Cat. # 3033; Cell Signaling Technology, Danvers, Massachusetts, USA; 1:1000), GAPDH (Cat. # 01404; StemCell Technologies, Vancouver, CA; 1:1000). The blots were developed using a chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
3
Visualizing Phosphorylated NF-κB Localization
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Transparent glass slides were placed at the bottom of six-well plates, coated with poly-D-lysine solution (Sangon Biotech, Shanghai, China) for 30 min, rinsed with phosphate-buffered saline, and dried. Cells (2 × 105 per well) were added and cultured with medium containing LPS for 6 h. The medium was aspirated, and cells were washed with PBS and fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China) before being washed three more times with PBS and incubated with 0.1% Triton X-100 (Sangon Biotech, Shanghai, China) for 15 min. After blocking with 3% bovine serum albumin (Sangon Biotech, Shanghai, China) for 1 h, cells were incubated with phospho-p65 antibody (#3033, Cell Signaling Technology, Danvers, MA, USA) for 1 h, washed three times, and incubated with Alexa Fluor 488 (Abcam, Cambridge, UK) for 1 h. Cells were then observed under a laser confocal microscope (Olympus FV1000, Tokyo, Japan).
4
Immunoblotting of Akt, p65 Signaling
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Anti-Akt (cat no. 4691), phospho-Akt (cat no. 4060), p65 (cat no. 6956), and phospho-p65 antibody (cat no. 76778) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-GAPDH (cat no. 014-25524), HRP-linked anti-mouse IgG (cat no. 115-035-146), and HRP-conjugated anti-mouse IgG (cat no. 115-035-003) antibody were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan).
5
Western Blot Analysis of Cell Signaling
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Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to NC membranes, and blocked with TBST of 5% skim milk for 1 hour. The membranes were washed with TBST and then incubated with the specific primary antibody for 6 hours at 4°C. The membranes were then incubated for 1 hour at room temperature in secondary and the signal was detected by chemiluminescence (Bio rad, USA). The primary (1:1000) and secondary (1:10000) antibodies were purchased from Cell Signaling Technology, USA and listed as follows: GAPDH antibody(#2118), stat3 antibody(#12640), phospho-stat3 antibody(#98543), SAPK/JNK antibody(#9252), phospho-SAPK/JNK antibody(#4668), p65 antibody(#4764), phospho-p65 antibody(#3033), IKKα antibody(#2682), phospho-IKKα/β antibody(#2697), IκBα antibody(#4812), phospho-IκBα antibody(#2859), p38 MAPK antibody(#8690), phospho-p38 MAPK antibody(#4511), Erk1/2 antibody(#4695), phospho-Erk1/2 antibody(#4370) and HRP-linked goat anti-rabbit IgG(#7074).
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