P p65 93h1

Manufactured by Cell Signaling Technology

Sourced in United States

P-p65 (93H1) is an antibody that recognizes phosphorylated p65, a subunit of the NF-κB transcription factor complex. This antibody can be used to detect and quantify the activated form of p65 in various experimental systems.

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3 protocols using p p65 93h1

Ginkgo biloba Extract Inhibits Inflammation

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Ginkgo biloba L. leaf extract injection (GBE) was purchased from Youcare Pharmaceutical Group (Beijing, China, Approval No. H20070226, Supporting Information 1). Its initial concentration is 3.5 mg/ml and prepared in aqueous solvent with excipients such as sorbitol, ethanol, sodium hydroxide. Lipopolysaccharide (LPS, #L2880) were obtained from sigma, and prepared in DMEM medium at concentration of 10 mg/ml. Primary antibodies for COX-2 (#4842, 1:1000), IκB-α (L35A5) (#4814, 1:1000), p-IκB-α (14D4) (#2859, 1:1000), NF-κB p65 (D14E12) (#8242, 1:1000) and p-p65 (93H1) (#3033, 1:1000), NF-κB p50 (D4P4D) (#13586, 1:1000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, United States). Primary antibodies for TNF-α (7B8A11) (#60291-1-Ig, 1:1000), IL-6 (#21865-1-AP, 1:1000), GAPDH (1E6D9) (#60004-1-Ig, 1:1000), β-actin (2D4H5) (#66009-1-Ig, 1:1000) and Lamin B1 (#12987-1-AP, 1:1000) were obtained from Proteintech Group Inc. (Rosemount, IL, United States). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Laboratories.

Liu M., Peng Y., Che Y., Zhou M., Bai Y., Tang W., Huang S., Zhang B., Deng S., Wang C, & Yu Z. (2022). MiR-146b-5p/TRAF6 axis is essential for Ginkgo biloba L. extract GBE to attenuate LPS-induced neuroinflammation. Frontiers in Pharmacology, 13, 978587.

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Ulinastatin Regulation of Osteoclastogenesis

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Ulinastatin was purchased from Techpool (Guangzhou, China), and dissolved in normal saline and serum-free α-MEM basic medium for using in vivo and in vitro, respectively. Recombinant soluble mouse RANKL and macrophage-colony stimulating factor (M-CSF) were obtained from Peprotech (Rocky Hill, CT, United States). Rabbit antibodies against p38(D13E1, #8690), p-p38(D3F9, #4511), ERK(137F5, #4695), p-ERK(D13.14.4E, #4370), JNK (#9252), p-JNK(81E11, #4668), p65(D14E12, #8242), p-p65(93H1, #3033), IκBα(L35A5, #4814), and p-IκBα(14D4, #2859) were acquired from Cell Signaling Technology (Boston, MA, United States). Mouse antibody against uPAR (ab103791) was obtained from Abcam (Cambridge, MA, United States). Mouse antibody against GAPDH (A00227-1, P04406) was obtained from Boster (Wuhan, China).

Huang J.M., Ren R.Y., Bao Y., Guo J.C., Xiang W., Jing X.Z., Shi J., Zhang G.X., Li L., Tian Y., Kang H, & Guo F.J. (2018). Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR. Frontiers in Pharmacology, 9, 1016.

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Multiparametric Flow Cytometric Analysis of Immune Cell Populations

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Mononuclear cell suspensions were prepared as previously described ^{10 (link),16 (link),17 (link)}. Antibodies for flow cytometry were purchased from eBioscience or BD Pharmingen and used at a concentration of 1:100 unless recommended otherwise by the manufacturer. Mouse AHR antibody (IC6697G) and mouse FLT-1 antibody (FAB4711A) were from R&D Systems, VEGF-B (RM0008–6E72) and TGF-α (MF9) from Novus Biologicals, EGF Receptor (D38B1) and p-p65 (93H1) from Cell Signaling. Cells were then analyzed on a LSRII or MACSQuant flow cytometer (BD Biosciences and Miltenyi Biotec, respectively). As outlined in the individual figures, Th1 cells were defined as CD3⁺CD4⁺IFN-γ⁺IL-17⁻IL-10⁻Foxp3⁻, Th17 cells as CD3⁺CD4⁺IFN-γ⁻IL-17⁺IL-10⁻Foxp3⁻, Treg cells as CD3⁺CD4⁺IFN-γ⁻IL-17⁻Foxp3⁺, microglia as CD11b⁺CD45^lowLy6C^low, and pro-inflammatory monocytes as CD45^hiCD11b⁺Ly6C^hi.

Rothhammer V., Borucki D.M., Tjon E.C., Takenaka M.C., Chao C.C., Fabregat A.A., de Lima K.A., Vazquez C.G., Hewson P., Staszewski O., Blain M., Healy L., Neziraj T., Borio M., Wheeler M., Dragin L.L., Laplaud D.A., Antel J., Alvarez J.I., Prinz M, & Quintana F.J. (2018). Microglial control of astrocytes in response to microbial metabolites. Nature, 557(7707), 724-728.

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