AR-C 155858 (AR-C) was from AdooQ Bioscience, α-Cyano-4-hydroxycinnamic acid (4-CIN) and isoproterenol from Sigma-Aldrich, and14C-lactate from PerkinElmer. Antibodies against MCT1 and MCT4 were from Millipore, and antibodies against carbonic anhydrase II (CAII) and NBCe1 from Abcam. Antibodies against p150Glued and transcription factor IIB (TFIIB) were from BD Biosciences and Santa Cruz Biotechnology, respectively. Unless otherwise mentioned, other reagents were purchased from Sigma-Aldrich. Antibodies against hormone-sensitive lipase (HSL), phospho-Ser660-HSL, phospho-Ser133 cAMP-responsive element binding protein (CREB), poly-ADP-ribose (PARP), and phospho-Thr180/Tyr182-p38 MAPK were from Cell Signaling Technology, Leiden, The Netherlands.
Phospho p38 mapk thr180 tyr182
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-p38 MAPK (Thr180/Tyr182) is a lab equipment product that detects the phosphorylation of p38 MAPK at Thr180 and Tyr182. This antibody can be used in various immunoassays to measure the activation of p38 MAPK.
Automatically generated - may contain errors
Lab products found in correlation
P38 mapk, by Cell Signaling Technology (28 mentions)
Sapk jnk, by Cell Signaling Technology (10 mentions)
Phospho sapk jnk thr183 tyr185, by Cell Signaling Technology (9 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Phospho akt ser473, by Cell Signaling Technology (7 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (6 mentions)
Phospho nf κb p65 ser536, by Cell Signaling Technology (5 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (5 mentions)
β actin, by Merck Group (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (4 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
Erk1 2, by Cell Signaling Technology (3 mentions)
Phospho histone h2a x ser139, by Cell Signaling Technology (3 mentions)
Total erk1 2, by Cell Signaling Technology (3 mentions)
Phospho akt thr308, by Cell Signaling Technology (3 mentions)
Phospho c jun ser73, by Cell Signaling Technology (3 mentions)
β actin, by Abcam (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Hsp27, by Cell Signaling Technology (2 mentions)
64 protocols using phospho p38 mapk thr180 tyr182
1
Comprehensive Cellular Metabolite Analysis
2
Endothelial Cell ERα and IκBα Regulation
3
Immunofluorescence Assay for Phospho-p38 MAPK
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Cells were extensively washed with PBS and fixed with 4% paraformaldehyde in Phosphate Buffer (PB) for 30 min in room temperature and washed 3 times with PBS. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 25 min at room temp, washed with PBS containing 0.2% Tween 20 (PBS-T), and blocked with FCS containing 10% (v/v) skin milk and 0.2% (v/v) tween 20 for 45 min. Cells were then incubated with Phospho-p38 MAPK (Thr180/Tyr182) (1:500; Cell Signaling) in PBS-T containing 10% skim milk for ON at 4°C. The Next day, cells were washed with PBS-T, and detection was performed using the fluorescent secondary antibody Alexa Fluor® 568 goat anti-rabbit IgG (1:500; Molecular Probes) for 45 min at room temperature. Finally, cells were washed with PBS-T and the coverslips were mounted in DAPI Fluoromount G (SouthenBiotech). Microscopic images were obtained using Olympus BX52 Fluorescence Microscope.
4
Quantifying Cellular Signaling Pathways
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All chemicals were purchased from either Sigma‐Aldrich (Poole, UK) or Fisher Scientific UK Ltd (Loughborough, UK), except where statedotherwise. Antibodies against phospho‐Akt (Ser473), phospho‐Akt (Thr308), Akt, phospho‐4EBP‐1 (Thr37/46), 4EBP‐1, phospho‐eIF2α (Ser51), eIF2α, phospho‐p38 MAPK (Thr180/Tyr182), p38 kinase, phospho‐AMP‐activating kinase(AMPK)α(Thr172), AMPKα, phospho‐heat shock protein (HSP)27 (Ser82) and HSP27 were obtained from Cell Signalling Technology (NEB, Hitchin, UK). Antibodies against XBP‐1, GRP94 and HSP60 were obtained from Abcam (Cambridge, UK), and HSP90 and HSP70 were obtained from Enzo Life Science (Exeter, UK). Anti‐GRP78 was from Transduction Laboratories (BD Biosciences, Oxford, UK) and anti‐β‐actin was obtained from Sigma‐Aldrich. OxPhos Complex Kit for mitochondrial electron transport chain (ETC) subunits was obtained from Invitrogen (Renfrew, UK).
5
Immunofluorescence Staining of Cells
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Cells were fixed with 4% paraformaldehyde (10 min at 37 °C) and permeabilized with 100% room-temperature methanol for 3 min. Cells were stained with the following primary human antibodies overnight at 4 °C: VE-Cadherin (1:80, Cayman Chemical); CD34 (1:80, BD Pharmingen); CD31 (1:80, BD Pharmingen); CD45 (1:80, BD Pharmingen); and phospho-p38 MAPK (Thr180/Tyr182, 1:100, Cell Signalling). Secondary staining was carried out using Alexa Fluor antibodies (1:200) incubated for 1 h at room temperature and protected from light. DAPI was used to visualize DNA content. Cells were imaged using the Cellomics Arrayscan VTI platform as previously described57 (link). Colony analysis, pattern fidelity and spatial organization of cells was performed using in-house clustering algorithms (content explorer) as previously described57 (link).
6
Cell Signaling Pathway Reagent Procurement
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Most chemicals, including dexamethasone, 3-isobutyl-1-methylxanthine, insulin, saponin, cycloheximide (CHX), verteporfin, crystal violet, anti-FLAG M2 affinity gels, Dulbecco's modified Eagle medium (DMEM), and antibodies against vinculin (V4505), β-actin (A2228), α-tubulin (T5168), and FLAG-tag (F1804) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Opti-MEM, Lipofectamine 2000, Lipofectamine RNAiMAX, bovine serum albumin, goat serum, 4′,6-diamidino-2-phenylindole (DAPI), and Alexa Fluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against PIP5Kα (#9693), PIP5Kγ (#3296), phospho-YAP (Ser127; #4911), YAP (#4912), phospho-LATS1 (Ser909; #9157), LATS1(#9153), phospho-TAZ (Ser89; #59971), TAZ (#4883), Merlin (#6995), HA-tag (#3724), Myc-tag (#2278 and #2276), p44/42 mitogen-activated protein kinase (MAPK, #4695), phospho-p44/42 MAPK (Thr202/Tyr204; #8544), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182; #9211), c-Jun N-terminal kinase (JNK, #9258), phospho-JNK (Thr183/Tyr185; #4668), Akt (#9272), and phospho-Akt (Ser473; #9271) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against lamin B1 (sc-374015), GAPDH (sc-47724), and green fluorescent protein (GFP, sc-9996) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
7
Signaling Pathway Antibody Validation
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Antibodies used were phospho-IkBa (Ser32/36) (9246, Cell Signaling), phospho-SAPK/JNK (Thr183/Tyr185) (9251, Cell Signaling), SAPK/JNK (9252, Cell Signaling), IkBa (SC-371, Santa Cruz), phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), Phospho-p38 MAPK (Thr180/Tyr182) (9215, Cell Signaling), Phospho-NF-κB p65 (Ser536) (3033, Cell Signaling), tubulin (ab7291, Abcam), actin (AAN01-A, Cytoskeleton), BiP (610978, BD Biosciences), CHOP (MA1-250, ThermoFisher), SLC39A7 (19429-1-AP, Proteintech), TNFR1 (sc-8436, Santa Cruz), TRAIL-R1/DR4 (42533, Cell Signaling), RIPK1 (610458, BD Bioscience), RIPK3 (12107, Cell Signaling), GFP (sc-69779, Santa Cruz), FADD (610399, BD Biosciences), phospho-MLKL (Ser385) (ab187091, Abcam), phospho-RIPK1 (S166) (65746, Cell Signaling), phospho-RIPK3 (S227) (ab209384, abcam), cleaved Caspase-3 (Asp175) (9661, Cell Signaling), V5 (R960-25, Invitrogen or ab9116, abcam), PDIA2 (ab2792, abcam), TNIP1/ABIN-1 (4664, Cell Signaling), cIAP1/BIRC2 (7065, Cell Signaling), cIAP2/BIRC3 (3130, Cell Signaling) and LAMP1 (ab25630, abcam). The secondary antibodies used were goat anti-mouse HRP (115-035-003, Jackson ImmunoResearch), goat anti-rabbit HRP (111-035-003, Jackson ImmunoResearch), Alexa Fluor 680 goat anti-mouse (A-21057, Molecular probes) and IRDye 800 donkey anti-rabbit (611-732-127, Rockland).
8
Western Blot Analysis of Cell Signaling
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Proteins from total cell lysates or tumor tissues were separated by standard SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were washed, blocked and incubated with primary antibodies against p44/42 MAPK (Erk1/2, #4695), phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, #4370), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182, #9211), SAPK/JNK (#9258), phospho-SAPK/JNK (Thr183/Tyr185, #9211), caspase-3 (#9665), cleaved PARP (Asp214, #9544) and PARP (#9542) all purchased from Cell Signaling Technology (Boston, MA, USA). The primary antibody against Dragon (AF3597, AF3630), and the anti-sheep (HAF016) or anti-goat (HAF109) secondary antibodies were purchased from R&D Systems (Minneapolis, MN, USA).
9
Western Blot Analysis of Protein Expression
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Protein concentrations were measured using the Pierce BCA protein assay kit. An equal amount of protein lysates was loaded onto 4–20% SDS-PAGE gels. Following electrophoresis, proteins were transferred to PVDF membranes. For detection of proteins, the membranes were incubated with the following antibodies overnight at 4 °C: SOD2 (A-2, Santa Cruz: sc-133134, 1:500 dilution); β-tubulin (9F3, Cell Signaling Technology: 2128, 1:1,000 dilution), ATP5A (Abcam: ab14748, 1:1000 dilution), β-actin (Thermo: AM4302, 1:10,000 dilution), HuR/ELAVL1 (3A2, Santa Cruz: sc-5261, 1:500 dilution), Phospho-p38 MAPK (Thr180/Tyr182, Cell Signaling Technology: 9211, 1:1000 dilution), p38 MAPK (A-12, Santa Cruz Biotechnology: sc-7972, 1:1000 dilution), MAPKAPK-2 (Cell signaling technology: 3042, 1:1000 dilution), Cox2/PTGS2 (D5H5, Cell Signaling: 12282, 1:1000 dilution). The blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo: 34096) after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Biosciences) for 1 h at RT.
10
Myricetin Modulates MAPK and NF-κB Signaling
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Myricetin (≥98%) purchased from Herbpurify (Chengdu, China) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) under sterile conditions to make a stocking solution of 40 mg/ml. The T-PER Tissue Protein Extraction Reagents and Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA) were used for total protein extraction and the measurement of total protein concentration, respectively. The antibodies used in this study were as follows: polyclonal rabbit anti-ERK1/2 (Proteintech), phospho-ERK1/2 (Thr202/Thr204) (Arigo), JNK (Proteintech), phospho-SAPK/JNK (Thr183 (221)+Thr185 223) (Arigo), p38 MAPK (Cell Signaling Technology), phospho-p38MAPK (Thr180/Tyr182) (Cell Signaling Technology), NF-κB p65 (Cell Signaling Technology), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology), IKK alpha, phospho-IKK alpha (Thr23) (Arigo), and monoclonal mouse anti β-actin (Proteintech) for inflammation reaction analysis and Staphylococcal α-toxin (Sigma-Aldrich) and HRP-conjugated secondary antibodies (Proteintech) for Hla production analysis. All of these antibodies were used as recommended by the manufacturers.
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