U937 and RAW264.7 cells were infected with BCG (MOI 1/5) and cells in the BCG + Rv2346c groups were treated with Rv2346c (500 pg/ml). After incubating for 24, 48, or 72 h, the cells were collected and lysed. The lysates were centrifuged, denatured, applied to an SDS-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were then blocked with 5% non-fat dry milk at room temperature for 1 h, followed by an incubation with primary antibodies at 4 °C overnight. The primary antibodies included an anti-NF-κB p65 (Abcam, MA, USA), an anti-NF-κB p65 (phospho S536) antibody (Abcam), an anti-p38 antibody (Abcam), an anti-p38 (phospho Y182) antibody (Abcam), and an anti-glyceraldehyde-phosphate dehydrogenase (GAPDH) antibody (Abcam). After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. The results were visualized via chemiluminescence using an ECL kit (Thermo scientific) and photographed with a Tanon MultiImager. The density of the immunoreactive bands was measured using ImageJ; GAPDH was used as an internal control.
Anti nf κb p65 phospho s536 antibody
Manufactured by Abcam
Sourced in United States
Anti-NF-κB p65 (phospho S536) antibody is a primary antibody that recognizes the phosphorylated form of the NF-κB p65 subunit at serine 536. This antibody can be used to detect and quantify the activated form of NF-κB p65 in various cellular and experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Anti p38 antibody, by Abcam (1 mentions)
Anti p38 phospho y182 antibody, by Abcam (1 mentions)
Anti nf κb p65, by Abcam (1 mentions)
Dako envision system hrp, by Agilent Technologies (1 mentions)
Dako liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Anti caspase 3 antibody, by Abcam (1 mentions)
Anti bax antibody, by Abcam (1 mentions)
Anti bcl 2 antibody, by Abcam (1 mentions)
Anti nf κb p65 antibody, by Abcam (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Nucleoprotein and cytoplasmic protein extraction kit, by Keygen Biotech (1 mentions)
4 protocols using anti nf κb p65 phospho s536 antibody
1
Investigating NF-κB and p38 Pathways in BCG-Infected Cells
2
Karacoline Modulates NF-κB Signaling
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Karacoline (PLC≥98% by high-performance liquid chromatography [HPLC]) was purchased from Shanghai Chengshao Biological (Shanghai, China). Anti-nuclear factor (NF)-κB p65 (acetyl K310) antibody and anti–NF–κB p65 (phospho S536) antibody were purchased from Abcam (Cambridge, MA, USA) while other antibodies were purchased from SAB (Baltimore, MD, USA). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from BBI (Markham, CA, USA). Recombinant rat TNF-α was purchased from Sangon Biotech (Shanghai, China). Rat MMP-14 ELISA Kit, Rat Col II ELISA Kit and Rat Aggrecan ELISA Kit were purchased from Enzyme-linked Biotechnology (Shanghai, China).
3
Immunohistochemical Analysis of AID, LMP1, and NF-κB
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Two percent paraformaldehyde-fixed PBMCs were washed by phosphate-buffered solution (PBS) and permeabilized by 0.5% Tween20/PBS for 10 min at room temperature. After washing by distilled water (DW), PBMCs were smeared on silane-coated slide, washed twice by DW, and air dried.
The proteins of AID, LMP1, and human NF-κB p65 were detected using IHC with the following primary antibodies and Dako EnVision+ System/HRP (Dako) and Dako Liquid DAB+ Substrate Chromogen System (Dako).
AID: anti-AID mouse IgG1-κ (Invitrogen), dilution 1: 200
LMP1: anti-EBV-LMP mouse IgG (Dako), dilution 1: 100
NF-κB: anti-NF-κB p65 (phospho S536) antibody (Abcam, Cambridge, United Kingdom), dilution 1: 100
We semiquantified the positive cell percentage,
+1: 10% and under
+2: over 10% and under 50%
+3: 50% and over
Concerning the positivity of NF-κB, we adopted only nuclear staining, because nuclear expression of NF-κB should be activated form, while cytoplasmic expression would be inactive form.
The proteins of AID, LMP1, and human NF-κB p65 were detected using IHC with the following primary antibodies and Dako EnVision+ System/HRP (Dako) and Dako Liquid DAB+ Substrate Chromogen System (Dako).
AID: anti-AID mouse IgG1-κ (Invitrogen), dilution 1: 200
LMP1: anti-EBV-LMP mouse IgG (Dako), dilution 1: 100
NF-κB: anti-NF-κB p65 (phospho S536) antibody (Abcam, Cambridge, United Kingdom), dilution 1: 100
We semiquantified the positive cell percentage,
+1: 10% and under
+2: over 10% and under 50%
+3: 50% and over
Concerning the positivity of NF-κB, we adopted only nuclear staining, because nuclear expression of NF-κB should be activated form, while cytoplasmic expression would be inactive form.
4
Western Blot Analysis of Apoptosis and NF-kB Signaling
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The total protein was extracted with the nucleoprotein and cytoplasmic protein extraction kit (Jiangsu Keygen Biotech Co., Ltd. China) and quantified by the BCA protein assay kit (Beijing Bomaide Gene Technology Co., Ltd., China). After that, the protein solution was subjected to electrophoresis, detached via SDS-PAGE (12% gels), and transferred to PVDF membranes. The PVDF membranes were blocked with 5% skim milk powder in TBST (Tris buffered saline with 0.5% Tween 20) for 2 h, and then incubated with primary antibodies overnight at 4° C. After washing with TBST for 5 times, the PVDF membranes were incubated with secondary antibody at room temperature for an hour. The protein bands were exposed by enhanced chemiluminescent (ECL) substrate kit (Labgic Technology Co., Ltd. Hefei, China) and analyzed by ImageJ software. The Anti-Bcl-2 antibody, Anti-Caspase-3 antibody, Anti-Bax antibody, Anti-NF-κB p65 antibody and Anti-NF-κB p65 (phospho S536) antibody were purchased from Abcam. The p38 MAPK, Phospho-p38 MAPK (Thr180/Tyr182), P44/42 MAPK (Erk1/2), and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) were purchased from Cell Signaling Technology. β-actin was severed as the internal control and anti-β-actin was purchased from Labgic Technology Co., Ltd.
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