LPS and Griess reagent (modified) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse TNF-α ELISA kit and IL-6 ELISA kit were purchased from eBioscience (San Diego, CA, USA). The antibody COX2, iNOS, phos-P65 (Ser536), P65, phos-TBK1, TBK1, phos-AKT (Ser473), AKT, phos-S6K (Thr389), S6K, phos–P38 (Thr180/Tyr182), P38, phos–JNK(Thr183/Tyr185), JNK, GAPDH (Cell Signaling, Danvers, MA, USA) and phos–ERK (Tyr204), ERK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), HRP-linked anti-mouse and anti-rabbit antibodies were obtained from Santa Cruz. SYBR Select Master Mix for RT-PCR amplification was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Primers for TNF-α, IL-6, iNOS and GAPDH were from Invitrogen Life Technologies (Carlsbad, CA, USA).
Phos p65
Manufactured by Cell Signaling Technology
Sourced in United States
Phos-P65 is a product from Cell Signaling Technology that is used to detect the phosphorylated form of the NF-κB p65 subunit. It is a specific antibody that can be used in various immunoassay techniques to quantify the levels of phosphorylated p65 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Hif 1α, by Proteintech (2 mentions)
Enhanced chemiluminescence kit, by Applygen (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Modified griess reagent, by Merck Group (1 mentions)
Mouse tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 6 elisa kit, by Thermo Fisher Scientific (1 mentions)
Phos akt, by Cell Signaling Technology (1 mentions)
Phos s6k, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Polyvinylidene uoride membrane, by Merck Group (1 mentions)
4 protocols using phos p65
1
Investigating Inflammatory Signaling Pathways
2
Evaluating Intestinal Barrier Integrity
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The expression of junction proteins (ZO1, occludin) and the overall expression and phosphorylation level of proteins in NFκB signaling were detected by Western blotting. The frozen jejunum tissue lysed in cold radioimmunoprecipitation assay buffer containing a protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA). Proteins (15 ng each) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in Tris-buffered saline, the membranes were incubated with primary and secondary antibodies and visualized using enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA) with the Viber Fusion FX7 imaging system (Viber 7 Lourmat, PAR, France). Antibodies used for ZO1 (1:500) and occludin (1:500) were obtained from Servicebio (Wuhan, China). TLR4 (1:500) antibody was purchased from Bioss (Beijing, China). IKKβ (1:1000), phos-IKKβ (1:1000), IKBα (1:1000), P65 (1:1000), phos-P65 (1:1000), and β-actin (1:1000) antibodies were purchased from Cell Signaling (Danvers, MA, USA).
3
Western Blot Analysis of HIF-1α, NF-κB Signaling
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PDLCs were collected and lysed in radioimmunoprecipitation assay buffer. Protein content was determined using a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes (Merck Millipore, Germany). After blocking, the membranes were incubated overnight at 4 °C in the presence of primary antibodies against HIF-1α (Proteintech Group, USA), phos-p65 (Cell Signaling Technology, USA), p65 (Abcam, Cambridge, UK), IκBα (Proteintech), and β-actin (ZSGB-Biotech, Beijing, China) at 1:1,000 dilution. After washing with TBST, the membranes were incubated with the corresponding secondary antibodies (1:10,000; ZSGB-Biotech) at room temperature for 1 h. The bands were visualized by a Bio-Rad system (ChemiDocTM MP Imaging System, USA) using an enhanced chemiluminescence kit (Applygen, Beijing, China). ImageJ software was used to quantify band intensities, the signals of which were normalized to that of β-actin or GAPDH.
4
Measuring Transcription Factor Activation
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PDLCs were collected and lysed in radioimmunoprecipitation assay buffer. Protein content was determined using a BCA kit (Thermo Fisher Scienti c, Waltham, MA, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene uoride membranes (Merck Millipore, Germany). After blocking, the membranes were incubated overnight at 4°C in the presence of primary antibodies against HIF-1α (Proteintech Group, USA), phos-p65 (Cell Signaling Technology, USA), p65 (Abcam, Cambridge,UK), IκBα (Proteintech), and β-actin (ZSGB-Biotech, Beijing, China) at 1:1,000 dilution. After washing with TBST, the membranes were incubated with the corresponding secondary antibodies (1:10000; ZSGB-Biotech) at room temperature for 1 h. The bands were visualized by a Bio-Rad system (ChemiDocTM MP Imaging System, USA) using an enhanced chemiluminescence kit (Applygen, Beijing, China). ImageJ software was used to quantify band intensities, the signals of which were normalized to that of β-actin or GAPDH.
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