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.
Anti phospho jnk thr183 tyr185
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-JNK (Thr183/Tyr185) is a laboratory reagent that detects the phosphorylated form of the c-Jun N-terminal kinase (JNK) protein. It can be used to monitor the activation state of JNK in various cellular and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Anti jnk, by Cell Signaling Technology (6 mentions)
Anti phospho p38 thr180 tyr182, by Cell Signaling Technology (4 mentions)
Anti p38 mapk, by Cell Signaling Technology (3 mentions)
Anti p38, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti phospho p65 ser536, by Cell Signaling Technology (2 mentions)
Hybond p polyvinylidene difluoride membrane, by Cytiva (2 mentions)
Anti p44 42 mapk, by Cell Signaling Technology (2 mentions)
Sheep anti mouse igg, by Cytiva (2 mentions)
Thrombin, by Chrono-Log (2 mentions)
U46619, by Chrono-Log (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Anti phospho acc ser79, by Cell Signaling Technology (2 mentions)
Anti phospho ampkα thr172, by Cell Signaling Technology (2 mentions)
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Anti phospho egfr tyr1068, by Cell Signaling Technology (1 mentions)
Anti phospho c fos ser32, by Cell Signaling Technology (1 mentions)
Minute total protein extraction kit for animal cultured cells tissues, by Invent Biotechnologies (1 mentions)
Sodium fluorescein, by Merck Group (1 mentions)
19 protocols using anti phospho jnk thr183 tyr185
1
Candida Infection of Vaginal Epithelial Cells
2
Licochalcone A Modulates Platelet Activation
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Licochalcone A (LA, ≥95%) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Collagen, thrombin, and U46619 were purchased from Chrono-Log (Havertown, PA, USA). FITC-conjugated anti-P-selectin and PAC-1 antibodies were purchased from Biolegend (San Diego, CA, USA). FITC-conjugated Collagen, phorbol-12, 13-dibutyrate (PDBu), luciferase/luciferin, fluorescein sodium, and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma (St. Louis, MO, USA). Fura 2-AM was purchased from Molecular Probe (Eugene, OR, USA). Anti-phospho PLCγ2 (Tyr759), anti-PLCγ2, anti-phospho-(Ser) PKC substrate, anti-phospho-p38 MAPK (Ser180/Tyr182), anti-phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204), anti-c-Jun N-terminal kinase (JNK), and anti-phospho-Akt (Ser473) polyclonal antibodies and anti-p38 MAPK, anti-p44/42 MAPK, anti-phospho JNK (Thr183/Tyr185), and anti-Akt monoclonal antibodies were purchased from Cell Signaling (Beverly, MA, USA). The pleckstrin (p47) antibody was purchased from GeneTex (Irvine, CA, USA). The Hybond-P polyvinylidene difluoride membrane, an enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system, horseradish peroxidase (HRP)-conjugated donkey antirabbit IgG, and sheep antimouse IgG were purchased from Amersham (Buckinghamshire, UK). LA was dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C until use.
3
Western Blot Analysis of Inflammatory Signaling
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Cells were plated at 0.3 × 106 cell/mL (low density) or 1.2 × 106 cell/mL (high density) were rinsed with cold PBS and lysed in 1% NP40 buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor (PhosSTOP, Roche). Cell lysates and supernatants were resolved in 12% acrylamide SDS-PAGE gels and blotted into a PVDF membrane for mIL-1β (H-153, Santa Cruz, Dallas, TX, USA), NLRP3 (Cryo-2 AG-20B-0014, Adipogen, Liestal Switzerland) or anti MAPK antibodies anti-Phospho JNK (Thr183,Tyr 185, Cat. 9251S), anti-JNK (Cat. 9252S), anti-Phospho-p44/42 (phospho-Erk1/2) (Thr202, Tyr 204, Cat. 4377), anti-p44/42 (Erk1/2), anti-phospho p38 (Thr 180/Tyr 185, Cat. 9211) or anti-p38 (Cat. 9212) (all from Cell Signaling Technology, Danvers, MA, USA). Primary antibody incubation was performed overnight in 3% bovine serum albumin (Sigma-Aldrich) or 5% w/v Difco skim milk (BD Biosciences). Primary antibodies were revealed using the corresponding secondary anti-mouse, or anti-rabbit IgG-peroxidase horseradish linked (GE-Healthcare, Munich, Germany). Analysis of protein bands was performed using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA), and values were normalized to β-actin.
4
Western Blot Analysis of Cellular Signaling
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Whole cell extraction was performed using RIPA buffer supplemented with protease inhibitors.
Samples were denatured in sodium dodecyl sulfate (SDS) sample buffer. Total proteins were separated by loading 20 μg of total cell lysate onto a denaturing SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 3% non-fat dry milk in phosphate-buffered saline containing 0.1% Triton X-100, followed by incubation with primary antibodies that recognize anti-Ero1l (Santa Cruz Biotechnology, Dallas, TX), anti-Akt, anti–phospho-Akt (Ser473), anti-JNK, anti–phospho-JNK (Thr183/Tyr185), anti–glyceraldehyde 3-phosphate dehydrogenase, and actin (Cell Signaling, Danvers, MD). Secondary antibody conjugated to horseradish peroxidase (Vector Laboratories Inc.) was used for detection of primary antibodies, and enzymatic signals were visualized using a chemiluminescence kit (Thermo Scientific). Western blots were quantitated using ImageJ software.
Samples were denatured in sodium dodecyl sulfate (SDS) sample buffer. Total proteins were separated by loading 20 μg of total cell lysate onto a denaturing SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 3% non-fat dry milk in phosphate-buffered saline containing 0.1% Triton X-100, followed by incubation with primary antibodies that recognize anti-Ero1l (Santa Cruz Biotechnology, Dallas, TX), anti-Akt, anti–phospho-Akt (Ser473), anti-JNK, anti–phospho-JNK (Thr183/Tyr185), anti–glyceraldehyde 3-phosphate dehydrogenase, and actin (Cell Signaling, Danvers, MD). Secondary antibody conjugated to horseradish peroxidase (Vector Laboratories Inc.) was used for detection of primary antibodies, and enzymatic signals were visualized using a chemiluminescence kit (Thermo Scientific). Western blots were quantitated using ImageJ software.
5
Immunohistochemical Analysis of Drosophila Wing Discs
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Standard protocols were used to fix and stain wing discs for immunohistochemistry. TO-PRO-3 or DAPI was used for a DNA counterstain (Invitrogen, Carlsbad, CA), and tissues were mounted in Vectashield (Vector Labs, Burlingame, CA). The following antibodies were used: anti-dpERK (1:100; Sigma-Aldrich, St. Louis, MO), anti-phospho Drosophila Akt (Ser505) (1:25; Cell Signaling, Danvers, MA), anti-Sima (1:100; generated by PRF&L, Canadensis, PA), anti-phospho PDH (S293) (1:100; Abcam, Cambridge, MA), anti-PDHK1 (1:100; Abcam), anti-phospho PDHK1 (Tyr243) (1:200; Cell Signaling), anti-phospho JNK (Thr183/Tyr185) (1:500; Cell Signaling), and anti-phospho Src (Tyr418) (1:200; Invitrogen). Single- plane fluorescence images were obtained using a Zeiss LSM 700 confocal microscope with Zen 2009 acquisition software. Images and average intensity of images were processed and measured using ImageJ. At least 20 discs were used for each genotype per experiment. Three technical replications were done for each experiment.
6
Antibody and Reagent Specifications for Cell Signaling Experiments
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Puromycin and G-418 were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). AG1478, and U0126 were purchased from Selleck. The protein A/G agarose and EGF were purchased from Sigma (St louis, MO, USA). The following antibodies were used: anti-β-actin (#8457), anti-cleaved-caspase-3 (#9664), ani-caspase-3 (#9662), anti-cleaved PARP (#5625), anti-EGFR (#4267), anti-Erk1/2 (#4695), anti-phospho-Erk1/2 (#4370), anti-p38 (#8690), anti-phospho-p38 (#9211), anti-AKT(#9272), anti-phospho-AKT (Ser473) (#4060), anti-phospho-JNK (Thr183/Tyr185) (#9251), anti-JNK (#9252), anti-HA-Tag (#3724), anti-c-Cbl (#2747), anti-ubiquitin (P4D1) (#3936) from Cell Signaling Technology (Beverly, MA, USA); anti-Flag-Tag from Sigma-Aldrich (St Louis, MO, USA), anti-GAPDH (AC002), anti-phospho-EGFR (Y1068) (AP0820) from Abclonal Technology(Shanghai, China); anti-14-3-3σ (ab14123), anti-Bim (ab7888) from Abcam (Cambridge, MA, USA); anti-Bcl-2 (12789-1-AP) from proteintech (Chicago, IL, USA); Alexa Flour 488-conjugated anti- rabbit IgG, Alexa Flour 555-conjugated anti-rabbit IgG and Alexa Flour 555-conjugated anti-mouse IgG from Thermo Scientific (Rockford, IL, USA). HRP conjugated anti-rabbit IgG, HRP conjugated anti-mouse IgG from Beyotime Institute of Biotechnology; APC anti-human EGFR antibody (#352905) used for cell surface immunofluorescence staining from BioLegend (San Diego, CA).
7
Antibodies for Cellular Signaling Analysis
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The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK). Anti-IGFIR antibody was a gift of S. Pons (CSIC, Spain).
8
AMPK Activation in HEK293 Cells
9
Protein Quantification and Western Blot Analysis
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Total proteins from cell lines and tissues were extracted in a lysis buffer (CAT.78510) (Thermo Fisher Scientific, Rockford, IL) and quantified using Bradford method (CAT.23226) (Thermo Fisher Scientific). Sixty micrograms of protein were separated by SDS-PAGE (10%). After transferring by Trans-Blo ® TurboTM (Bio-Rad, USA), the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies Mig-6 (1: 1000, CAT.11630-1-AP) (protein tech), anti-P62/SQSTM1 (1: 1000,CAT.55274-1-AP)(protein tech), GFP (1:1000,CAT.ab38689) (Abcam), β-actin (1:2000, CAT.20536-1-AP) (protein tech), anti-phospho-JNK (Thr183/Tyr185) (1:1000, CAT.4668) (Cell Signaling Technology, Danvers, MA), anti-LC3b (1:2000, CAT.NB100-2220) (Novus Biologicals USA), anti-JNK (1:1000, CAT.AJ518) (Beyotime Biotechnology). After incubation with peroxidase-coupled anti-mouse or rabbit IgG (Santa Cruz Biotechnology) at 37°C for 2 hours, bound proteins were visualized using ECL (Thermo Fisher Scientific) and detected using ChemidocTM MP Imaging Systerm (Bio-Rad, USA). The relative protein levels were calculated based on β-actin as the loading control.
10
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).
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