Proteins were extracted from the cells using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 5 mM phenylmethylsulfonyl fluoride, and 1 mM DTT) containing 1% NP-40. Lysates were centrifuged at 10,000× g for 10 min at 4 °C. Protein concentrations were determined using a bicinchoninic acid protein assay (Sigma, Steinheim, Germany) according to the manufacturer’s instructions. Twenty micrograms of protein were separated by electrophoresis using 10% SDS–polyacrylamide gel and then transferred to polyvinylidene fluoride membranes (Amersham, UK). Membranes were subsequently blocked with 1% nonfat dry milk in Tris-buffered saline Tween 20 (TBST) at room temperature and incubated with p-JNK antibody (Cell Signaling Technology, Beverly, MA, USA) with 1% bovine serum albumin in TBST overnight at 4 °C, followed by incubation for 60 min at room temperature with HRP-conjugated secondary antibody. Proteins were visualized by enhanced chemiluminescence (Amersham, UK).
P jnk antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The P-JNK antibody is a laboratory reagent used to detect the phosphorylated form of the c-Jun N-terminal kinase (JNK) protein. JNK is a member of the mitogen-activated protein kinase (MAPK) family and plays a role in cellular signaling pathways. The P-JNK antibody specifically binds to and identifies the phosphorylated, active form of JNK, which can be used to study the activation of this protein in various experimental systems.
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Lab products found in correlation
Jnk antibody, by Cell Signaling Technology (3 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Bicinchoninic acid protein assay, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Anti mouse igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti p p38 antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti p akt antibody, by Cell Signaling Technology (1 mentions)
Anti p erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Calyculin a, by Abcam (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sp600125, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
U0126, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti mrp1, by Santa Cruz Biotechnology (1 mentions)
5 cfda, by Merck Group (1 mentions)
Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
8 protocols using p jnk antibody
1
Protein Extraction and Western Blot Analysis
2
Activation and Characterization of JNK3
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WT JNK3 39–402 and JNK3 39–402 L144I were activated with upstream kinases MKK4 and MKK7, which were purchased from Millipore. 300 nM of JNK3 proteins in 200 μL volume were incubated for 2 h at 30°C with 100 nM each of MKK4 and MKK7 in kinase reaction buffer containing 25 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM DTT, 1 mM β-pyrophosphate and 200 μM ATP. The phosphorylation state of the activated JNK3 proteins was confirmed by mass spectrometry analysis, Western analysis using p-JNK antibody, (Cell Signaling) and specific activity assays.
3
Fibroblast Signaling Pathway Analysis
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The primary fibroblasts were treated with TGFβ, FGF, PDGF (7666-MB, 3139-FB, 1447-PC, R&D Systems), or PP1 inhibitor Calyculin A (141784, Abcam) for 15 or 30 min, respectively. After 2 times of PBS washing, the total protein was extracted by lysate with protease inhibitor and phosphatase inhibitor. The total protein concentration was detected by BCA kit, and the protein was separated by 10% SDS-PAGE and then transferred to PVDF membrane. The 5% skimmed milk was used to block the protein at room temperature for 2 h, and then the primary antibodies were added and reacted at 4 °C overnight. On the second day, the corresponding second antibody was added and sealed at room temperature for 1 h, followed by the final step of ECL addition for exposure. The antibodies contain anti-p-Smad2/3 antibody (8828, Cell Signaling Technology, 1:1 K), anti-p-P38 antibody (9211, Cell Signaling Technology, 1:1 K), anti-p-ERK1/2 antibody (4370, Cell Signaling Technology, 1:2 K), anti-p-AKT antibody (4060, Cell Signaling Technology, 1:2 K), p-JNK antibody (4668, Cell Signaling Technology, 1:1 K), anti-GAPDH antibody (30201ES, Yeasen, China, 1:1 W), anti-rabbit IgG secondary antibody (305-035-003, Jackson ImmunoResearch, 1:1 W) and anti-mouse IgG secondary antibody (115-035-003, Jackson ImmunoResearch, 1:5 K).
4
Investigating AITC-induced Cellular Responses
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AITC was purchased from Anhui Haibei Import and Export Company (Hefei, Anhui, China). RPMI 1640 medium and Fetal Bovine Serum were purchased from Gibco. LY294002, U0126, SP600125, 5-CFDA, and sodium dodecyl sulfate (SDS) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Monoclonal antibodies, including anti-MRP1, β-actin, and JNK, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-JNK antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA).
5
Anti-Inflammatory Effects Evaluation
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The cell line—macrophage cell (RAW 264.7)—used for the measurement of cell viability was purchased from the Korean Cell Line Bank (Seoul, Korea). The reagents for cell culture, fetal bovine serum (FBS), and Dulbecco’s modified eagle medium (DMEM) were purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA). The reagents used for anti-inflammatory measurement experiments, MTT, Griess reagent, RIPA lysis and extraction buffer, LPS, protease inhibitor, phosphatase inhibitor, and nuclear and cytoplasmic extraction reagents were purchased from Sigma Aldrich Co., Ltd. The iNOS antibody, COX−2 antibody, donkey anti-mouse IgG-HRP, and mouse anti-rabbit IgG-HRP for the Western blot analysis were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). JNK antibody, p-JNK antibody, ERK1/2 antibody, p-ERK1/2 antibody, and NF-kB (p65) antibody were obtained from Cell Signaling Technology (Beverly, MA, USA).
6
Estrogen-Induced Oxidative Stress Response
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OVX rat injected E2 (Tocris Bioscience, Bristol, UK; catalog no. 2824), Estradiol ELISA kit (Biovision, Milpitas, CA, USA; catalog no. K7417-100). The primary antibodies used for western blot and immunofluorescence staining were 8-OHDG antibody (Abcam, Cambridge, UK; catalog no. ab48508), BiP antibody (Thermo Fisher Scientific, Lafayette, Colorado; catalog no. PA1-014A), Sigma receptor 1 antibody (Santa Cruz, California, USA; catalog no. sc-137075), pJNK antibody (Cell Signaling Technology, Boston, Massachusetts, USA; catalog no. 4668), JNK antibody (Cell Signaling Technology; catalog no. 9252), PDI antibody (Cell Signaling Technology; catalog no.3501), Ero-1α antibody (Cell Signaling Technology; catalog no.3264), Calnexin antibody (Cell Signaling Technology; catalog no.2679) and β-actin (Cell Signaling Technology; catalog no. 8457). The secondary antibodies used for western blot and immunofluorescence staining were the anti-rabbit lgG, HRP linked antibody (Cell Signaling Technology; catalog no. 7074), anti-mouse lgG, HRP linked antibody (Cell Signaling Technology; catalog no. 7076), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Thermo Fisher Scientific; catalog no. A32728) and Goat anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific; catalog no. A-11029).
7
Immunofluorescence Analysis of Signaling Proteins
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For immunofluorescence investigations, cells grown on glass coverslips were fixed for 30 minutes at room temperature in 4% paraformaldehyde in 0.1 M phosphate buffer (30) . Cell membranes were permeabilized by incubation for 30 minutes in 0.05 M Tris buffer (pH 7.4) containing 0.1% Triton X-100, 2% BSA, and 2% normal goat serum. Cultures were incubated overnight at 4℃ in primary antibody solution containing either phosphor-APP (Thr668) antibody (1:500, Cell Signaling), MAP2 antibody (1:500, EMD Millipore), p-JNK antibody (1:500, Cell Signaling), JIP-1 antibody (1:50, Santa Cruz), or JIP-3 antibody (1:50, Santa Cruz). Cells were then washed, followed by incubation with fluorescein-isothiocyanate (FITC)- or Cy3-labeled secondary antiserum (1:300, Jackson Laboratories).
8
Icariin and Ferric Ammonium Citrate Protocol
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Icariin (United States, 489-32-7) and ferric ammonium citrate (FAC, United States, 1185-57-5) were purchased from Sigma-Aldrich (Supplementary Figure S1 ). Cleaved caspase-3 (Asp175) antibody (United States, #9661), MFN2 antibody (United States, #11925), DRP1 antibody (United States, #8570), COX IV antibody (United States, #4850), RUNX-2 antibody (United States, #12556), active β-catenin antibody (United States, #19807), cyclin D1 antibody (United States, #2922), PI3K antibody (United States, #4257), P-PI3K antibody (United States, #4228), AkT antibody (United States, #4691), P-AkT antibody (United States, #4060), mTOR antibody (United States, #2972), P-mTOR antibody (United States, #5536), P-ERK antibody (United States, #4370), ERK antibody (United States, #4695), P-p38 antibody (United States, #4511), p38 antibody (United States, #8690), P-JNK antibody (United States, #4668), JNK antibody (United States, #9252) were all purchased from Cell Signaling Technology. BAX antibody (United States, 50599-2-Ig), FIS1 antibody (United States, 10956-1-AP), cytochrome C antibody (United States, 66264-1-Ig) and osteopontin (OPN) antibody (United States, 22952-1-AP) were all purchased from Proteintech (United States, 50599-2-Ig). Bcl-2 antibody was purchased from R&D system (United States, MAB8272). Anti-beta actin antibody was purchased from BOSTER (China, BM3873).
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