Cells were harvested and lysed in RIPA buffer containing a protease inhibitor cocktail (P3100; GenDEPOT, Katy, TX, USA). Blots were probed using anti-c-Raf (1:1000, Cell Signaling Technologies, 9422S), anti-phosphoAkt (Ser473) (1:1000, Cell signaling, 9271S), anti-Akt(1:1000, Cell signaling, 9272S), anti-phospho-p44/42 MAPK (Thr202/Tyr204) (1:1000, Cell signaling, 9101S), anti-p44/42 MAPK (1:1000, Cell signaling, 9102S), anti–phosphoMEK 1/2 (1:1000, Cell Signaling Technologies, 2338S), and anti-β-actin (1:3000, sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).
Anti p44 42 mapk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p44/42 MAPK is a laboratory equipment product that detects the expression of the p44/42 MAPK (Erk1/2) protein. It is a primary antibody that specifically recognizes the p44/42 MAPK proteins and can be used in various cell and tissue-based applications to analyze the activation and cellular localization of this important signaling molecule.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho p44 42 mapk, by Cell Signaling Technology (18 mentions)
Anti p38 mapk, by Cell Signaling Technology (7 mentions)
Anti akt, by Cell Signaling Technology (6 mentions)
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti phospho p44 42 mapk thr202 tyr204, by Cell Signaling Technology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti mouse igg, by Bio-Rad (2 mentions)
Anti 4e bp1, by Cell Signaling Technology (2 mentions)
Modified bradford assay, by Bio-Rad (2 mentions)
Anti egfr, by Cell Signaling Technology (2 mentions)
Goat anti rabbit igg, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Hybond p polyvinylidene difluoride membrane, by Cytiva (2 mentions)
Sheep anti mouse igg, by Cytiva (2 mentions)
Anti phospho jnk thr183 tyr185, by Cell Signaling Technology (2 mentions)
Thrombin, by Chrono-Log (2 mentions)
36 protocols using anti p44 42 mapk
1
Immunoblotting Analysis of Signaling Pathways
2
Oli-neuM Cell Culture and Immunoblot
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Typically, 2.75 × 105 Oli-neuM cells were seeded in 6-well plates in GM media and cells were grown to 70% confluence. Treatments, unless otherwise specified, were performed as indicated in the text, as previously described. For immunoblot analyses, the following antibodies were used: Cell Signaling Technology (Danvers, MA, USA): anti-phospho-p44/42 MAPK (Erk1/2Thr202/Thr204: #9101, 1:5000), anti-p44/42 MAPK (Erk1/2: #1240, 1:3000), anti-Phospho AKT (Ser473: #4060, 1:2500); anti-AKT (pan: #4691, 1:2000). Sigma-Aldrich: anti-actin (2066, 1:2000). AbD Serotec (Bio-Rad Laboratories, Hercules, CA, USA): anti-MBP (MCA409S, 1:200). Proteintech® (Proteintech Group, Rosemont, IL, USA): anti-RXRγ (11129-1-AP,1:600). GeneTex (GeneTex, Inc., Irvine, CA, USA): anti-EGFR (GTX132810, 1:1000). Cell extract (CE) preparation and immunoblot analyses were performed as previously described [8 (link)]; briefly, bands signal intensity was estimated using ImageJ software (version 1.8.0), and data were plotted using GraphPad Prism 7.0 (GrahPad Software, San Diego, CA, USA) as the fold change versus vehicle, arbitrarily set to 1.
3
Multiplex Analysis of Signaling Proteins
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Protein lysates containing equal amount of proteins, measured by a modified Bradford assay (BIORAD, Hercules, CA), were subjected to direct Western Blot (WB). Immuno-complexes were dectected with the enhanced chemiluminescence kit (ECL plus, Thermo Fisher Scientific, Rockford, IL). We used the following antibodies from Cell Signalling (Beverly, MA): anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-HER2, anti-phospho-HER2 (Tyr1248), anti-HER3, anti-phospho-HER3 (Tyr1289), anti-IGF1R-beta, anti-phospho-IGF1R-beta (Tyr1135), anti-p44/42 MAPK, anti-phospho-p44/42MAPK, anti-AKT, anti-phospho-AKT (Ser 473), anti-AXL, anti-c-MET, anti-S6 ribosomal protein, anti-phospho-S6 ribosomal protein, anti-4EBP1, anti-phospho-4EBP1, anti-vimentin, anti-E-cadherin, anti-Snail. Anti-α-tubulin (internal loading control) was from Sigma (Sigma-Aldrich, St. Louis, MO). The following secondary antibodies from Biorad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG. Each experiment was done in triplicate.
4
Protein Expression Profiling in hPSC-derived AECs
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Protein extracted from hPSC-derived AECs were lysed in protein lysis buffer and quantified using the bicinchoninic acid protein assay. The 20 μg of protein was separated by SDS-PAGE using 8–15% gel and then transferred to polyvinylidene fluoride membranes. Nonspecific binding proteins were blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated with primary antibodies against anti-phospho-p44/42 mitogen-activated protein kinase (Cell Signaling Technology, Danvers, USA, 4370), anti-p44/42 MAPK (Cell Signaling Technology, 4695), anti-SFTPC (Abcam, ab40879), anti-ACE2 (R&D, Minneapoli, MN, USA, AF933), and anti-fibronectin (FN) (Santa Cruz Biotechnology, sc-59826) overnight at 4 °C. Membranes were scanned with ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
5
Immunoprecipitation and Immunoblotting Analyses
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Immunoprecipitation and immunoblotting analyses were performed as previously described [33 (link)]. The following primary antibodies were used for immunoblotting: rabbit anti-CD93 (H190) and mouse anti-c-Myc (9E10, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-β-actin (Sigma-Aldrich, St Louis, MO, USA); anti-p44/42 MAPK and anti-phospho-p44/42 MAPK (Cell Signaling, Danvers, MA, USA). For immunoprecipitations, the protein extracts were incubated for 2 h at 4° C with the mAb 4E1 coupled to Dynabeads® Pan Mouse IgG (Invitrogen).
6
Western Blot Analysis of Cellular Signaling
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Cells were lysed using lysis buffer and centrifuged at 10000 r.p.m. for 30 minutes at 4°C. The supernatant was collected, and the total protein concentration was quantified using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The protein extracts were heated for 10 min at 95°C and NuPAGE 4-12% Bis-tris gel (Invitrogen) was used to load protein samples for electrophoresis. After the electrophoresis, protein membranes were incubated with anti-phospho-p44/42 MAPK (Erk1/2), anti-p44/42 MAPK (ErK1/2)), anti-AKT antibody, anti-phospho-AKT, anti-STAT3 antibody, anti-phospho-STAT3 antibody (all from Cell Signalling, Massachusetts, USA) or anti-GAPDH antibody (Sigma-Aldrich, Missouri, USA) overnight at 4°C. The membrane was then incubated with anti-rabbit IgG, HRP linked antibody (Cell signalling) for 1hr. The membrane was visualized through enhanced chemiluminescence system (Santa Cruz). Protein bands were captured by Syngene GeneSnap software (Syngene, Cambridge, UK) and analyzed by Image J (NIH, Bethesda, USA).
7
Immunoblotting with Antibody Panel
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The antibodies used in this study were as follows: anti-EGFR (D38B1) rabbit mAb (4267), anti–HA-tag (6E2) mouse mAb (2367), anti-EGFR (D38B1) rabbit mAb (4267), anti-insulin receptor β (L55B10) mouse mAb (3020), anti–IGF-I receptor β (D23H3) rabbit mAb (9750), anti-MET (D1C2) rabbit mAb, anti-FER (5D2) mouse mAb (8198), anti-RAB5 (C8B1) rabbit mAb (3547), anti-RAB11 (D4F5) rabbit mAb (5589), anti-RAB7 (D95F2)XP(R) rabbit mAb (9367), anti–phospho-p44/42 MAPK (T202/Y204) rabbit mAb (9101), and anti-p44/42 MAPK (Erk1/2; L34F12) mouse mAb (4696); (all from Cell Signaling Technology); anti-Vinculin (SPM227) mouse mAb (ab18058; Abcam), anti–EGFR-ECD mouse mAb (Ab-3, clone EGFR.1; ms311; Thermo Fisher Scientific), anti-PKCδ mouse mAb (610398; BD Transduction Laboratories), anti-RAB7 (D-4) mouse mAb (sc-271608, IF; Santa Cruz Biotechnology), and anti-myc (9E10 clone) mouse mAb (generous gift of Dr. Timothy Hercus, Centre for Cancer Biology, an alliance of SA Pathology and University of South Australia, Adelaide, Australia).
8
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.
9
Regulation of C/EBPβ and MAPK Signaling in U937 Cells
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U937 cells were cultured in standard conditions with fatty acids (24 hours, 100 µM) or 5-aza-dC (daily additions for 2 days, 1 µM f.c.). Protein samples from total cell lysates (50 µg) were subjected to SDS-polyacrilamide gel electrophoresis, electroblotted onto a nitrocellulose membrane (Schleicher and Schuell), and probed using the following antibodies: anti-phospho-C/EBPβ (Thr235) #3084, anti-phospho-p44/42 MAPK (pERK1/2, T-202/Y-204) #9101, anti-p44/42 MAPK #9102, anti-pan-Ras #3965 (Cell Signaling). Anti-H-Ras specific antibody 18295-1-AP (Proteintech), anti-N-Ras (F155:sc-31) and anti-K-Ras (F234:sc-30) (Santa Cruz Biotechnology). Immunoreactive bands were visualized using the ECL assay (Amersham Pharmacia Biotech, Amersham). Anti-β-tubulin antibody (Sigma-Aldrich) was used to normalize. Images were acquired using the VersaDoc Imaging System (Bio-Rad), and signals were quantified using Quantity One software (Bio-Rad).
10
Signaling Pathways in 6-OHDA-Induced Toxicity
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SH-SY5Y cells (1.5 × 106 cells) were differentiated, pre-treated with 0.5% or 1% BJ for 1 h and then incubated with 50-µM 6-OHDA for additional 6 or 24 h. Total cellular lysates were prepared, quantified and electrophoresed (30 µg/lane) as reported by Celano et al. [32 (link)]. The membranes were blocked with 5% nonfat milk and then incubated overnight at 4 °C with the following antibodies: rabbit anti-phospho p44/42 MAPK (Thr 202/Try 204) and anti-p44/42 MAPK (Cell Signaling Technology, Beverly, MA, USA); rabbit anti-phospho p38 and rabbit anti-p38 (AbCam); rabbit anti-iNOS and nNOS (Becton Dickinson, Franklin Lakes, NJ, USA); mouse anti-p53 (AbCam); mouse anti-Bax and anti-Bcl-2 (Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-β-actin (Cell Signaling Technology). Secondary goat HRP-conjugated anti-mouse or anti-rabbit IgG antibody (AbCam) were incubated at room temperature for 2 h. A representative immunoblot image of three independent experiments for each target is shown. Autoradiographic bands were quantified by ImageJ software and normalized for β-actin levels.
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