The rhizomes of S. glabra were purchased from Kangmei Pharmaceutical Co. Ltd. (Guangdong, China) in February 2013 (batch No. 12120527), and were verified by Huang Zhi-hai in the Second Institute of Clinical Medicine, Guangzhou University of Chinese medicine (Guangzhou, China). The samples were air-dried at 45 °C, and then pulverized to pass through 100 mesh sieves to obtain fine powder. Astilbin was purchased from Chengdu Must Bio-technology Co. (Chengdu, China) with the purity more than 98% in high performance liquid chromatography (HPLC) analysis. The RAW264.7 cell line was obtained from the Laboratory Animal Center, Sun Yat-Sen University (Guangzhou, China). Dulbeccos’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were produced by Invitrogen-Gibco (Grand Island, NY, USA). MTT, LPS and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse TNF-α and IL-6 enzyme-linked immune sorbent assay (ELISA) kits were purchased from R & D Systems, Inc (St. Louis, MO, USA). The anti-phosphorylated NF-κB P65, anti-phosphorylated JNK, anti-phosphorylated ERK1/2, and anti-phosphorylated p38 mouse or rabbit antibodies were purchased from Cell Signaling Technology (Danvers, CO, USA).
Anti phosphorylated nf κb p65
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-phosphorylated-NF-κB p65 is a primary antibody that specifically recognizes the p65 subunit of NF-κB when it is phosphorylated. This antibody can be used to detect the activation and nuclear translocation of NF-κB, a transcription factor involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti phosphorylated jnk, by Cell Signaling Technology (2 mentions)
Anti tata, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti traf6, by Santa Cruz Biotechnology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Anti nf κb p65, by Cell Signaling Technology (2 mentions)
Anti phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Astilbin, by Chengdu Must Bio-Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti cyclophilin b, by Abcam (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Fujifilm (1 mentions)
Anti socs3, by Cell Signaling Technology (1 mentions)
Nitrocellulose filter paper sandwiches, by Bio-Rad (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey system, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit igg secondary antibody, by LI COR (1 mentions)
Anti phosphorylated p38, by Cell Signaling Technology (1 mentions)
8 protocols using anti phosphorylated nf κb p65
1
Astilbin Modulates Inflammatory Response
2
Protein Expression Analysis in Cell Lines
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Western blot analysis of protein expressions in RAW264.7 and TCMK-1 cell lines was performed as previously described.17 (link) The primary antibodies used were as follows: anti-CX3CL1 (DF12376), anti-ARG-1 (DF6657), anti-ERK (AF0155), and anti-phosphorylated-ERK (AF1015) were purchased from Affinity Biosciences, Cincinnati, OH. Anti-CX3CR1 (13885-1-AP), anti-TNF-α (60291-1-IG), anti-CD206 (60143-1-Ig) and anti-NF-κB p65 (10745-1-AP) were purchased from Proteintech Group Inc. (Rosemont, IL). The other antibodies used were as follows: anti-CD80 (bs-2211R, Bioss, Beijing, China), and anti-phosphorylated-NF-κB p65 (#3033, Cell Signaling Technology, Danvers, MA). GAPDH (AB-P-R001, Xianzhi Biotechnology, Hangzhou, China) was used for normalization.
3
Fetal Membrane NF-κB and MAPK Analysis
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Western blot analysis of NF-κB and MAPK was performed in the fetal membrane samples. Tissues were lysed in RIPA buffer (J63306: Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor (165-26021: Fuji film, Osaka, Japan), and 1000 μg of protein extracts were subjected to electrophoresis using 4–12% SDS-PAGE gels (NW04127: Thermo Fisher Scientific, Waltham, MA, USA). Proteins were transferred to membranes using an iBlot2 Dry Blotting System (IB21001: Thermo Fisher Scientific, Waltham, MA, USA). The membranes were incubated overnight with the following primary antibodies: anti-phosphorylated-NF-κB p65 (1:1000; #3033: Cell Signaling Technology (CST), Danvers, MA, USA), anti-NF-κB p65 (1:3000; #8242: CST), anti-phosphorylated-JNK (1:1000; #4668: CST), anti-JNK (1:1000; #9252: CST) anti-phosphorylated-p38 MAPK (1:3000; #4511: CST), anti-p38 MAPK (1:3000; #8690: CST), and anti-cyclophilin B (1:20,000; ab178397: Abcam, Cambridge, UK). Cyclophilin B was used as a loading control. Protein bands were densitometrically analyzed using Image Saver 5 for the Ez-Capture series (ATTO Corporation, Tokyo, Japan). Image J version 1.51 was used to calculate the relative densities of the bands (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Axl and Signaling
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the lysates (30 μg/lane) of lung and cell culture protein, which were then transferred to a polyvinylidene fluoride membrane (Millipore). The blots were incubated overnight with primary antibodies, including anti-Axl, anti- phosphorylated-Axl (1:1000, Bioss Inc., Beijing, China), anti-TNF receptor associated factor 6 (anti-TRAF6) (1:200, Santa Cruz Biotechnology, USA), anti-SOCS3, anti-phosphorylated-NF-κB p65, anti-IκB-α, anti-TATA (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:10000, Sigma Chemical Company, USA).
5
Western Blot Analysis of Phosphorylated NF-κB
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Isolated protein samples were run on a 4–15% protein gel in Tris/Glycine/SDS buffer (BIO-RAD) at 180 V for 30 min and transferred onto 0.2 µm Nitrocellulose filter paper sandwiches (BIO-RAD) in 100 mM CAPS and 20% methanol at < 8 °C and 30 V for 2 h. Membranes were then rinsed with Tween-20/TTBS and blocked in Odyssey Blocking buffer (LI-COR) for 1 h. Primary antibody labeling was carried out with anti-phosphorylated NFκB p65 and anti- NFκB p65 (Cell Signaling 3031S and 4764S, respectively) (diluted to 1:1000; 88 ng/ml in Odyssey Blocking buffer) on a shaker overnight at 4˚C. Membranes were rinsed four times with TTBS for 15 min each, and then labeled with IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR) (diluted to 1:10,000 in Odyssey Blocking buffer) on a shaker in the dark at RT for 1 h. Membranes were rinsed with TTBS again, and air-dried in the dark. Final blotting results were resolved using a LI-COR Odyssey system. (Full-length blot image for gel bands shown in Fig. 2 B is shown in Supplemental Figure S8 .)
6
Lung Protein Expression Analysis
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The lysates (30 μg/lane) of lung and cell culture protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore). The blots were incubated overnight with primary antibodies anti-α-ENaC, anti-TRAF6 (1:200, Santa Cruz Biotechnology, USA), anti-phosphorylated-p38, anti-p38, anti-phosphorylated-Erk, anti-Erk, anti-phosphorylated-JNK, anti-JNK, anti-phosphorylated-NF-κB p65, anti-IκB-α, anti-TATA (1:1,000, Cell Signaling Technology, USA), and anti-β-actin (1:10,000, Sigma Chemical Company, USA). The anti-phosphorylated NKCC1 and anti-NKCC1 antibodies were custom made by GeneTex.
7
Exosome and Kidney Tissue Protein Analysis
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The protein lysates from exosomes or kidney tissues were prepared following standard protocols, and the protein content was determined using the BCA protein assay kit. Then, protein samples were separated by Bis-Tris Gel (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% BSA in TBST for 1 h at room temperature and were incubated with primary antibodies overnight at 4 °C. Then, membranes were washed three times and incubated with secondary antibodies for 2 h at room temperature, and the signals were detected using an ECL advanced system (GE Healthcare). Intensity values expressed as the relative protein expression were normalized to GAPDH (AB2000, Abways). Primary antibodies used were anti-Alix [35 (link)] (sc-53540, Santa), anti-CD63 (ab193349, Abcam), anti-CD9 (ab92726, Abcam), and anti-phosphorylated NF-κB p65 (3033, Cell Signaling Technology). Secondary HRP-conjugated antibodies used were anti-mouse IgG and anti-rabbit IgG (Abcam).
8
Western Blot Analysis of NF-κB and STAT3
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Western blot analysis was performed using a modified method described previously [23 (link)]. Each antibody was used at the following dilutions:
anti-phosphorylated-NF-κB/p65 (product no. 3033), 1:1,000 dilution; anti-NF-κB/p65 (product no. 3034), 1:1,000 dilution; anti-phosphorylated-STAT3 (product no. 9131), 1:1,000 dilution;
anti-STAT3 (product no. 9139); Cell Signaling Technology, Danvers, MA, USA), 1:1,000 dilution; and anti-β-actin (code no. PM053; Medical & Biological Laboratories Co., Ltd.), 1:1,000
dilution. Then, the membrane was exposed to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (product no. 7074; Cell Signaling Technology). Chemiluminescence signals were
obtained from reactions with Chemi Lumi One Plus Reagent (Nacalai Tesque), and data were obtained using an LAS4000 system (Fujifilm, Tokyo, Japan). β-actin was used as an internal control to
normalize differences in the amount of protein appliqued.
anti-phosphorylated-NF-κB/p65 (product no. 3033), 1:1,000 dilution; anti-NF-κB/p65 (product no. 3034), 1:1,000 dilution; anti-phosphorylated-STAT3 (product no. 9131), 1:1,000 dilution;
anti-STAT3 (product no. 9139); Cell Signaling Technology, Danvers, MA, USA), 1:1,000 dilution; and anti-β-actin (code no. PM053; Medical & Biological Laboratories Co., Ltd.), 1:1,000
dilution. Then, the membrane was exposed to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (product no. 7074; Cell Signaling Technology). Chemiluminescence signals were
obtained from reactions with Chemi Lumi One Plus Reagent (Nacalai Tesque), and data were obtained using an LAS4000 system (Fujifilm, Tokyo, Japan). β-actin was used as an internal control to
normalize differences in the amount of protein appliqued.
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