Protein from co-cultured cells was extracted using the protease inhibitor cocktail and phosphatase inhibitor cocktail (Kangchen Bio-tech, China). Cell lysates were subjected to SDS-PAGE electrophoresis and proteins were blotted onto PVDF membranes. Appropriate primary antibodies and corresponding HRP-conjugated secondary antibodies were used. Exposure was performed using enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA) and x-ray film. Antibodies used in western blotting were: anti-p38, anti-phospho-Thr180/Tyr182 p38, anti-ERK1/2, anti-phospho-Thr202/Tyr204 ERK1/2, anti-JNK, anti-phospho-Thr183/Tyr185 SAPK/JNK, and anti-GAPDH (all from Cell Signaling Technology).
Anti phospho p38 thr180 tyr182
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-phospho-p38 (Thr180/Tyr182) is a laboratory reagent used to detect the phosphorylation of p38 MAPK at threonine 180 and tyrosine 182 residues. This antibody can be used in various immunoassay techniques to measure the activation of p38 MAPK signaling pathways.
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Lab products found in correlation
Anti p38, by Cell Signaling Technology (11 mentions)
Anti erk1 2, by Cell Signaling Technology (5 mentions)
Anti phospho jnk thr183 tyr185, by Cell Signaling Technology (4 mentions)
Anti jnk, by Cell Signaling Technology (3 mentions)
Anti phospho erk thr202 tyr204, by Cell Signaling Technology (3 mentions)
Anti erk, by Cell Signaling Technology (3 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti phospho p65 ser536, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Kangchen Biotech (1 mentions)
Phosphatase inhibitor cocktail, by Kangchen Biotech (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Anti phospho thr202 tyr204 erk 1 2, by Cell Signaling Technology (1 mentions)
Cell culture dishes, by BD (1 mentions)
Anti jnk1 2, by Cell Signaling Technology (1 mentions)
Sb203580, by Merck Group (1 mentions)
Cell culture medium, by BD (1 mentions)
Anti nox4 sc 30141, by Santa Cruz Biotechnology (1 mentions)
18 protocols using anti phospho p38 thr180 tyr182
1
Western Blot Analysis of Kinase Activation
2
Inflammatory Pathway Modulation Protocol
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(-)-EC (>95% of purity), DHBA (≥99% of purity), DHPAA (>98% of purity), HPPA (>98% of purity), diphenyleneiodonium (DPI), and SB203580 were purchased from Sigma Chemicals (Madrid, Spain). Lipopolysaccharide (LPS) from the E. coli 0111:B4 strain was obtained from Invivogen (Nucliber, Madrid, Spain). TNF-α and IL-6 kits were acquired from R&D Systems (Abingdon, UK) and Invitrogen (Thermo Fisher, Madrid, Spain), respectively. Anti-ERK1/2 and anti-phospho-ERK1/2 recognizing phosphorylated Thr202/Thy204 of ERK1/2, anti-JNK1/2 and anti-phospho-JNK1/2 recognizing phosphorylated Thr183/Tyr185 of JNK1/2, anti-phospho-Thr180/Tyr182-p38, and anti-β-actin were obtained from Cell Signalling Technology (Izasa, Madrid, Spain). Anti-TNF-α (sc-52746), anti-IL-6 (sc-57315), anti-VCAM-1 (sc-13160), anti-ICAM-1 (sc-107), anti-MCP-1 (sc-52701), p38α (sc-535), and anti-NOX-4 (sc-30141) were purchased from Santa Cruz Biotechnology (Quimigen, Madrid, Spain). Materials and chemicals for electrophoresis were from BioRad (BioRad Laboratories S.A., Madrid, Spain). Cell culture dishes, glutamine and cell culture medium were from Falcon (Cajal, Madrid, Spain) and Lonza (Madrid, Spain), respectively.
3
Candida Infection of Vaginal Epithelial Cells
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The VK2/E6E7 vaginal epithelial cells were seeded onto six-well tissue culture plates and incubated in supplement-free KSFM for 12 h and then infected with Candida cells with a multiplicity of infection (MOI) of 5. EGFR inhibitors were added to the host cells 2h before the fungal stimulation. At various time points, the epithelial cells were rinsed with cold PBS and lysed using a modified RIPA lysis buffer containing protease (Cell Signaling Technology) and phosphatase (Sigma-Aldrich) inhibitors, left on ice for 30 min. The cells were collected by centrifugation and supernatants were assayed for total protein. 20 ug of the protein was separated by SDS-PAGE and the proteins were detected by immunoblotting with specific antibodies, including anti-phospho-EGFR Tyr1068 (#3777), anti-phospho-c-Fos Ser32 (Cell signaling; #5348), anti-phospho-p65 Ser536 (Cell signaling; #3033), anti-phospho-JNK Thr183/Tyr185 (Cell signaling; #9255),anti-phospho-Erk1/2 Thr202/Thr204 (Cell signaling; #4370), anti-phospho-p38 Thr180/Tyr182 (Cell signaling; #4511). For the extraction of total protein from vaginal tissue, half of the dissected vagina was firstly grinded and then lysed using Minute™ Total Protein Extraction Kit for Animal Cultured Cells/Tissues (SD-001/SN-002, invent biotechnologies, America) at 4°C. After quantitation, the tissue protein was separated by SDS-PAGE and detected as above.
4
Immunofluorescence Staining of Immune Cells
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Mouse monoclonal antibodies included; ED1 (CD68, macrophages) and R73 (rat αβ T‐cell receptor) (Serotec, Oxford, UK), RP1 (neutrophils) (Becton Dickinson, San Diego, USA) and anti‐α‐tubulin (Abcam, Cambridge, UK). Rabbit polyclonal antibodies included anti‐phospho‐p38 Thr180/Tyr182 and anti‐phospho‐c‐Jun Ser63 (Cell Signaling, Boston, MA, USA) and goat anti‐γ‐fibrinogen (Santa Cruz biotechnology, CA, USA). Biotinylated antibodies included goat anti‐mouse IgG and goat anti‐rabbit IgG (Zymed, San Francisco, CA, USA). Immunofluorescence staining used FITC (Fluorescein isothiocyanate)‐conjugated rabbit polyclonal antibodies against sheep IgG Dako, Glostrup, Denmark, rat IgG (Sigma‐Aldrich, Castle Hill, NSW, Australia) and rat C3 (Cappel, Malvern, PA, USA).
5
Investigating IQGAP3 and ERK Interaction
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Myc-IQGAP3 and HA-ERK1 or HA-ERK2 constructs were transfected into HEK293T cells. Cells were harvested and lysed in lysis buffer with proteinase inhibitor cocktail (Roche, Basel, Switzerland) and phenylmethylsulfonyl fluoride. Then the cell lysates were incubated with mouse anti-HA mAb (Sigma-Aldrich), anti-Myc mAb (Sigma-Aldrich) or a control antibody (mIgG) (Sigma-Aldrich) and protein-A Sepharose (GE Healthcare, USA) and resolved by SDS-PAGE. For endogenous immunoprecipitation, cell lysate was prepared from A549 cells and immunoprecipitated with rabbit anti-ERK1 mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
For western blot analysis, equal amounts of protein from each sample was loaded and resolved with SDS-PAGE and transferred to blots. After blocking, blots were probed with indicated antibodies and evaluated with the Odyssey Imaging System (LICOR Bioscience, Lincoln, NE, USA). Antibodies used included anti-GAPDH (Bioworld Technology, Inc., St Louis Park, MN, USA), anti-IQGAP3 (Sigma-Aldrich), anti-ERK (Cell Signaling, Beverly, MA, USA), anti-phospho-ErkThr202/Tyr204 (Cell Signaling), anti-phospho-p38Thr180/Tyr182 (Cell Signaling), anti-phospho-AktSer473 (Cell Signaling), anti-Myc, and anti-HA.
For western blot analysis, equal amounts of protein from each sample was loaded and resolved with SDS-PAGE and transferred to blots. After blocking, blots were probed with indicated antibodies and evaluated with the Odyssey Imaging System (LICOR Bioscience, Lincoln, NE, USA). Antibodies used included anti-GAPDH (Bioworld Technology, Inc., St Louis Park, MN, USA), anti-IQGAP3 (Sigma-Aldrich), anti-ERK (Cell Signaling, Beverly, MA, USA), anti-phospho-ErkThr202/Tyr204 (Cell Signaling), anti-phospho-p38Thr180/Tyr182 (Cell Signaling), anti-phospho-AktSer473 (Cell Signaling), anti-Myc, and anti-HA.
6
Immunoblotting and Chromatin Immunoprecipitation Assays
7
Cardiac Connexin43 Regulation in Rats
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The cardiac ventricles of the rat and the cultured H9c2 cells (Chinese Academy of Sciences Cell Bank, Shanghai) were homogenized on ice and lysed in RIPA buffer (P0013B Beyotime, China) supplemented with proteinase and phosphatase inhibitors. Each protein in the sample was separated by 10% SDS‐PAGE gels and transferred to the PVDF membrane (Millipore, USA). The membranes were blocked by 5% skim milk in PBS containing 0.05% Tween 20 for 1 hour and incubated with anti‐GAPDH antibody (Abcam, 1:5000), anti‐connexin43 (Sigma, 1:10000), anti‐phospho‐Cx43 (Ser368) (Cell Signalling, 1:1000), anti‐p38 (Cell Signalling, 1:1000) and anti‐phospho‐p38 (Thr180/Tyr182) (Cell Signalling, 1:1000) overnight at 4°C. Afterwards, the membranes were washed in PBS containing 0.05% Tween 20, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson, 1:10000) for 1 hour at room temperature. The immunoreactivity was visualized by an enhanced chemiluminescence (ECL) advanced kit (Millipore). Signal intensities were analysed using Image J software.
8
AMPK Activation in HEK293 Cells
9
Isolation and Characterization of Plitidepsin
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Plitidepsin (C57H87N7O15, MW:1109.6, CAS No. 137219-37-5, APL), [14C]-plitidepsin (1.73 GBq/mmol), and a fluorescent coumarinated plitidepsin derivative (plitidepsin-DMAC) were prepared by PharmaMar (Colmenar Viejo, Spain). Stock solutions (1 mg/ml in DMSO) were prepared and stored at −20 °C. Complete (protease) and PhosStop (phosphatase) inhibitor cocktails were purchased from Roche Diagnostics (Mannheim, Germany). Anti-phospho-JNK (Thr183/Tyr185), anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-phospho-p38 (Thr180/Tyr182) antibodies were purchased from Cell Signaling Technologies, Inc (Beverly, MA, USA). Anti-eEF1A and secondary HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-eEF1A2 (GTX102326) antibody was purchased from GeneTex (Irving, CA, USA). Anti-α-Tubulin (#T5168) antibody and subtilisin (EC 3.4.21.62) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). A plasmid encoding eEF1A2-GFP (RG210716) was purchased from Origene (Rockville, MD, USA). Chromatography media (DEAE FF 16/10, SP HiPrep 16/10 and Superdex 200 16/600 columns) were obtained from GE Healthcare (Buckinghamshire, UK). All other reagents were from Sigma (St Louis, MO, USA).
10
Antibody Procurement for Cell Signaling
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Antibodies were obtained as follows: anti-NFAT5 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-actin antibody was from Sigma (Deisenhofen, Germany); anti-phospho-GSK-3β (Ser9), anti-GSK-3β, anti-phospho-p38 (Thr180/Tyr182), and anti-p38 were purchased from Cell Signaling (Beverly, MA, USA); anti-phospho-Akt (Ser473) and anti-AKT were from Genscript (Piscataway, NJ, USA). GSK-3β inhibitor VIII was obtained from Cayman Chemical (Ann Arbor, MI, USA). Unless otherwise indicated, other reagents were purchased from Biomol (Hamburg, Germany), Biozol (Eching, Germany), Carl Roth (Karlsruhe, Germany), or Sigma.
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