The CMECs were homogenized in RIPA lysis buffer (#P0013C; Beyotime) containing 1X Phosphatase Inhibitor Cocktail (#5870S; Cell Signaling Technology, Beverly, MA, USA) and 1 µg/ml each of aprotinin (A1153; Sigma-Aldrich) and leupeptin (#L2884; Sigma-Aldrich). Forty micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and then probed with antibodies for XO (#ab109235; Abcam), phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199) (#4228), PI3 kinase p85 (19H8) (#4257), phospho-p38 MAP kinase (Thr180/Thr182) (#4631), p38 MAPK (D13E1) (#8690), JAK2 (D2E12) (#3230) and phospho-JAK2 (Tyr1007/1008) (C80C3) (#3776) (all from Cell Signaling Technology, Beverly, MA, USA). The same membranes were reprobed with an antibody for tubulin (#AT819; Beyotime). The blotting film was quantified using a scanner and a densitometry program (ImageJ; https://imagej.nih.gov/ij/index.html ) (24 ). To quantify the phosphor-specific signal in the activated samples, the background was subtracted and the band was normalized to the amount of tubulin or total target protein in the lysate.
Ab109235
Manufactured by Abcam
Sourced in United Kingdom
Ab109235 is a lab equipment product offered by Abcam. It is a device designed for use in scientific research and laboratory applications. The core function of this product is to [DESCRIPTION_NOT_AVAILABLE].
Automatically generated - may contain errors
Lab products found in correlation
Jak2 d2e12, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Phospho p38 map kinase, by Cell Signaling Technology (1 mentions)
Pi3 kinase p85 19h8, by Cell Signaling Technology (1 mentions)
P38mapk d13e1, by Cell Signaling Technology (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
A 21244, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Slowfade diamond, by Thermo Fisher Scientific (1 mentions)
Cm1950, by Leica camera (1 mentions)
A11004, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Ab108980, by Abcam (1 mentions)
Ab51952, by Abcam (1 mentions)
Amersham imager 600rgb, by GE Healthcare (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
6 protocols using ab109235
1
Analyzing Protein Signaling Cascades
2
Immunohistochemical analysis of TA muscle
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TA muscles were excised, embedded in OCT compound (23-730-571, Thermo Fisher Scientific), and frozen in liquid nitrogen–cooled 2-methylbutane. A cryostat (Leica CM 1950) was used to obtain 10 μm muscle sections onto microslides (48311-703, VWR). The slides were fixed in 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton X-100/1× PBS, and blocked in 0.5% BSA/10% goat serum/1× PBS for 20 minutes. The slides were incubated overnight with anti–xanthine oxidase antibody (1:100, rabbit monoclonal, ab109235, Abcam) and anti-CD31 antibody (1:100, mouse monoclonal, 66065-2-lg, Proteintech) in 0.5% BSA/2% goat serum/1× PBS. For the secondary antibodies, the slides were washed and incubated with goat anti-rabbit IgG (H+L) Alexa Fluor 647 and goat anti-mouse Alexa Fluor 568 (1:300, A-21244 and A-11004, Thermo Fisher Scientific) in the dark for 1 hour. The slides were washed and incubated with DAPI/1× PBS for 15 minutes to detect nuclei. Slides were mounted with SlowFade Diamond (S36967, Thermo Fisher Scientific) and imaged using a Keyence BZ-X910 at 20× original magnification. Image processing was done using ImageJ.
3
Expression Profiling of Oxidative Enzymes in Differentiating HSkMCs
4
Synovial Membrane Protein Analysis
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Total protein was isolated from synovial membrane tissue samples. Analysis of its protein content was performed by Western blot as previously reported [31 (link), 32 (link)]. Prolidase, Nox2, XO, and NALP3 antibodies from Abcam (Ab108980, Ab-5826, Ab109235, and Ab51952, resp.) were used. Loading normalization was performed by anti-actin antibody (Sigma, A3854). The blots were scanned with Amersham Imager 600 RGB (GE) and densitometry analysis was performed with ImageQuant TL 8.1 Software.
5
Western Blotting Analysis of Cell Signaling
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The western blotting protocol was adapted from Zollbrecht et al.29 (link) Proteins extracted from cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer with 1% protease inhibitor mixture (PIC) and 1% phenylmethylsulphonyl fluoride (PMSF). The samples were separated on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride membranes in a semi-dry transfer apparatus (4561033, Bio-Rad, Hercules, CA, USA). The membranes were pre-incubated at room temperature in 5% dehydrated milk for 2 hours. Then each membrane was incubated with specific primary antibodies against IKKα (2684, Cell Signaling Technology, Danvers, MA, USA), p-IKKα (2694, Cell Signaling Technology), NF-κB (p65) (3034, Cell Signaling Technology), p-NF-κB (p65) (3033, Cell Signaling Technology), iNOS (13120, Cell Signaling Technology), XOR (ab109235, Abcam, Cambridge, UK), and β-actin (AC026, Abclone, Wuhan, China) overnight. Next, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (AS014, Abclone) and visualized using enhanced chemiluminescence reagents (1705062, Bio-Rad).
6
Oxidative Stress and Inflammation Markers
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Primary antibodies against xanthine oxidase (#ab109235), phospho-H2A histone family member X (γH2AX; Ser139; #ab131382), hypoxia-inducible factor 1-alpha (HIF1α; #ab179483), and nuclear factor erythroid 2-related factor 2 (NRF2; #ab62352), were purchased from Abcam (Cambridge, U.K.). Antibodies to detect p53 (#9282), phospho-p53 (Ser15, #9284) and β-actin (#3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). Tumor necrosis factor-α (TNFα) and Interleukin 6 (IL-6) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). XOR activity, uric acid (UA) content, and hydrogen peroxide assay kits were purchased from Solarbio Science & Technology (Beijing, China).
Evaluating DNA damage using comet, reduced glutathione (GSH), and myeloperoxidase (MPO) assay kits were purchased from Jiancheng Technology (Nanjing, China). Allopurinol was purchased from Sigma-Aldrich (St. Louis, MO, USA), while diphenyleneiodonium sulfate (DPI) was purchased from Toronto Research Chemicals (Toronto, ON, Canada). The siRNA transfection reagent (sc-29528) used to transfect siRNA into cells was purchased from Santa Cruz Biotechnology. Dextran sulfate sodium (DSS) was purchased from MP Biomedicals. Azoxymethane (AOM), CoCl 2 , and other chemicals were provided by Sigma-Aldrich (St. Louis, MO, USA).
Evaluating DNA damage using comet, reduced glutathione (GSH), and myeloperoxidase (MPO) assay kits were purchased from Jiancheng Technology (Nanjing, China). Allopurinol was purchased from Sigma-Aldrich (St. Louis, MO, USA), while diphenyleneiodonium sulfate (DPI) was purchased from Toronto Research Chemicals (Toronto, ON, Canada). The siRNA transfection reagent (sc-29528) used to transfect siRNA into cells was purchased from Santa Cruz Biotechnology. Dextran sulfate sodium (DSS) was purchased from MP Biomedicals. Azoxymethane (AOM), CoCl 2 , and other chemicals were provided by Sigma-Aldrich (St. Louis, MO, USA).
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