The total proteins of THP-1 cells infected with Wt, ΔM5447, or Comp strain were prepared at 24 h post-infection, and the concentration of proteins was determined by a BCA Protein Quantification kit (Vazyme). After separation of proteins by SDS-PAGE, the proteins were transferred onto an NC membrane. The membrane was blocked with 5% (w/v) non-fat milk in TBST buffer for 2 h at room temperature and incubated with primary antibodies, NF-κB p65 (Cell Signal Technology, Danvers, MA, United States), pSer536-NF-κB p65 (Cell Signal Technology), or GAPDH (Proteintech), at 4°C overnight. After washing three times with TBST buffer, the membrane was incubated with appropriate HRP-conjugated secondary antibody (Beyotime Biotechnology) for 1 h at room temperature. Finally, the protein bands were visualized by Western Bright ECL detection reagents (Advansta) and quantified using ImageJ software.
P ser536 nfκb p65
Manufactured by Cell Signaling Technology
Sourced in United States
P-Ser536 NFκB p65 is a lab equipment product that detects the phosphorylation of the serine 536 residue on the NF-κB p65 subunit. This phosphorylation event is a key regulatory mechanism of NF-κB activation.
Automatically generated - may contain errors
Lab products found in correlation
Hsp90 h 114, by Santa Cruz Biotechnology (2 mentions)
Jnk1 2 sc 571, by Santa Cruz Biotechnology (2 mentions)
P thr202 tyr204 erk1 2, by Santa Cruz Biotechnology (2 mentions)
P mek1 2 ser217 221, by Cell Signaling Technology (2 mentions)
P thr183 tyr185 jnk1 2, by Cell Signaling Technology (2 mentions)
P ampkα thr172, by Cell Signaling Technology (2 mentions)
Erk1 2, by Santa Cruz Biotechnology (2 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
Mek1 2, by Cell Signaling Technology (2 mentions)
Ampkα, by Cell Signaling Technology (2 mentions)
Phosphatase inhibitor, by LC Laboratories (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Western bright ecl detection reagents, by Advansta (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Bca protein quantification kit, by Vazyme (1 mentions)
Gapdh, by Proteintech (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
3 protocols using p ser536 nfκb p65
1
Analysis of NF-κB Activation in THP-1 Cells
2
Western Blot Analysis of Signaling Pathways
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Preparation of whole cell lysates and Western blots were performed as previously described54 . Phosphatase Inhibitor (LC laboratories) was added to inhibit phosphatase activity. Antibodies used in this study were: HSP90 (H-114, 1:6,000) and JNK1/2 (sc-571, 1:2,000) from Santa Cruz; p-Thr202/Tyr204 ERK1/2 (4370, 1:2,000), ERK1/2 (9102, 1:2,000), p-Ser217/221 MEK1/2 (9121, 1:1,000), MEK1/2 (4694, 1:1,000), p-Thr183/Tyr185 JNK1/2 (9255S, 1:2,000,), p-Ser473 AKT (9271S, 1:2,000), AKT (9272, 1:2,000), IκBα (9242, 1:2,000), p-Ser536 NFκB p65 (3033, 1:1,000), p-Thr172 AMPKα (2535, 1:2,000) and AMPKα (2532, 1:2,000) from Cell Signaling. Band density was quantitated using the Image Lab software on the ChemiDOC XRS+ system (Bio-Rad). Protein levels were normalized to HSP90 and the phosphorylated forms were normalized to phosphorylation-independent levels of the same protein. Data are presented as mean ± SEM unless otherwise specified. Uncropped blots are shown in Supplementary Fig. 9 –11 .
3
Western Blot Analysis of Signaling Pathways
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