Cell lysates was cleared by centrifugation and the protein concentration of the supernatant was determined using a DC protein assay reagent (Bio-Rad Laboratories). Levels of phosphorylated and total proteins were analyzed by western blotting. For western blot analysis, anti-phospho-EGFR (pY1068), anti-ERK (p44/42 MAP kinase) and anti-phospho-ERK (Thr202/Tyr204) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-EGFR antibody was purchased from Fitzgerald Industries (North Acton, MA, USA). The protein band intensities were quantified using a densitometer (Fuji Film Corp., Tokyo, Japan or ImageQuant LAS4000; GE Healthcare, Milwaukee, WI, USA). In all of the reported results, error bars denote the SE for at least two independent experiments.
Anti phospho erk thr202 tyr204
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-ERK (Thr202/Tyr204) is a primary antibody that recognizes the phosphorylated form of extracellular signal-regulated kinase (ERK) at threonine 202 and tyrosine 204 residues.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk, by Cell Signaling Technology (9 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (4 mentions)
Anti phospho p38 thr180 tyr182, by Cell Signaling Technology (3 mentions)
Anti p38, by Cell Signaling Technology (3 mentions)
Dc protein assay reagent, by Bio-Rad (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Anti egfr antibody, by Biosynth (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Anti pan akt, by Cell Signaling Technology (1 mentions)
Laser confocal microscope, by Nikon (1 mentions)
Anti hsp60, by Santa Cruz Biotechnology (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Igg alexa fluor 594, by Cell Signaling Technology (1 mentions)
Igg alexa fluor 488, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Anti myc mab, by Merck Group (1 mentions)
Mouse anti ha mab, by Merck Group (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
19 protocols using anti phospho erk thr202 tyr204
1
EGFR and ERK Phosphorylation Analysis
2
Immunofluorescence Analysis of Kinase Signaling
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In addition to KTRs, we identified changes in active and total ERK and Akt kinases using immunofluorescence staining. We seeded cells as described in the “Kinase translocation reporter ” section and stained cells in response to serum at the time points indicated in the figure. We used the following primary antibodies from Cell Signaling Technologies (Danvers, MA, USA): anti-phospho-Akt (Ser473) (cat. 4058), anti-pan-Akt (cat. 2920), anti-phospho-ERK (Thr202/Tyr204) (cat. 4370), and anti-pan-ERK (cat. 4696). We detected primary antibodies with fluorescent secondary antibodies from Jackson ImmunoResearch Laboratores Inc. (West Grove, PA, USA): anti-rabbit AlexaFluor 488 (cat. 111-545-003) and anti-mouse AlexaFluor 594 (cat. 115-585-003).
3
Multimodal Mitochondrial Imaging and Protein Localization
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Cells were cultured on coverslips and incubated in DMEM containing 100 nM MitoTracker Red (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C in the dark. After staining, cells were washed with fresh growth medium, and fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 min. Cells were then permeabilized with PBS containing 0.2% Triton X-100 for 3 min at room temperature, and incubated overnight with anti-HSP60 (1:300; Santa Cruz Biotechnology) or anti-phospho-ERK (Thr202/Tyr204) (1:250; Cell Signaling Technology) antibodies at 4 °C in a darkroom. Next, cells were washed with PBS and incubated with a fluorescently labeled secondary antibody either IgG-Alexa Fluor 594 or IgG-Alexa Fluor 488 (1:300; Cell Signaling Technology) for 1 h at room temperature in the dark. A final incubation was performed to stain the cells with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, Jiangsu, China) for 15 min at room temperature. After mounting the coverslips, images were captured using a confocal laser microscope at a magnification of ×600 (Nikon, Telford, UK).
4
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.
5
Comprehensive Western Blot Analysis
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Western blotting was performed using the protocol as previously described [18 (link)]. The following antibodies were used: anti-β-actin, anti-AR (G-4) and anti-TGF-α (D-6) (Santa Cruz Biotechnology); anti-EGF (4E11) (Life Technologies); anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-phospho-JNK (Thr183/Tyr185), anti-JNK, anti-phospho-Erk (Thr202/Tyr204) and anti-Erk (Cell Signaling Technology). Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the optical density was analyzed using a UVP Bioimaging system (UVP, Upland, CA).
6
Protein Expression Analysis by Western Blot
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Treated and control cells were harvested in 60 μl of radio-immuno-precipitation assay (RIPA) lysis buffer. The lysate was centrifuged (13 000 rpm, 15 min, 4 °C), and the supernatant was stored at −80 °C. Equal amounts of total protein (30 μg) were electrophoresed on 12% or 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. The blots were probed overnight at 4 °C with an anti- FGFR2 (Cell Signaling Technology Cat# 23,328, RRID: AB_2,798,862), anti-actin (Sigma-Aldrich Cat# A3853, RRID: AB_262,137), anti-phospho-FGFR2 (ser 782) (Thermo Fisher Scientific Cat# PA5–64,796, RRID: AB_2,662,677), anti-phospho-Erk (Thr202/Tyr204) (Cell Signaling Technology Cat# 4370, RRID: AB_2,315,112) and anti-Erk (Abcam Cat# ab17942, RRID: AB_2,297,336) primary antibody diluted in PBST (DPBS 10X (GibcoTM) after 1X dilution; 0.1% Tween-20) with 5% bovine serum albumin. The horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc. Dallas, USA) was diluted 1:10,000. Bound antibodies were visualized on Fusion Fx7 imaging system (Fisher Bioblock Scientific, Waltham, USA) using the ImmobilonTM Western enhanced chemiluminescence detection kit (Millipore Corporation, Billerica, USA). The resulting bands were analyzed and quantified using ImageJ 1.48v software (RRID: SCR_003070, National Institutes of Health, Bethesda, USA).
7
Platelet Activation Pathway Analysis
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2-MeSADP, thrombin, apyrase, prostaglandin E1 (PGE1), sodium citrate, prednisolone, and acetylsalicylic acid (ASA) were purchased from Sigma (St. Louis, MO, USA). Anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-phospho-ERK (Thr202/Tyr204), and anti-total-ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-linked secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Thromboxane B2 (TxB2) ELISA kit was purchased from Enzo Life Sciences (Exeter, UK). All other reagents were of reagent grade.
8
Protein Extraction and Immunoblot Analysis of Organoids
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Organoids were collected using a cell recovery solution (Corning) and washed with PBS, and proteins were extracted using 1% NP‐40 buffer (20 mmol/L Tris‐HCl, pH 8.0, 137 mmol/L NaCl, 1% NP‐40, 10% glycerol). The protein concentrations were determined using a protein assay (Bio‐Rad, Hercules, CA, USA), and equal amounts of protein were resolved, transferred to nitrocellulose membranes and detected as previously described.11 The antibodies used for the immunoblot analysis were as follows: anti‐STAT1(D1K9Y, Cell Signaling), anti‐phosphoSTAT1 (Ser727) (D3B7, Cell Signaling, Danvers, MA, USA), anti‐phosphoSTAT1 (Tyr701) (58D6, Cell Signaling), anti‐ERK (137F5, Cell Signaling), anti‐phosphoERK (Thr202/Tyr204) (D13.14.4E, Cell Signaling), anti‐S6 (5G10, Cell Signaling), anti‐phosphoS6 (Ser235/236) (D57.2.2E, Cell Signaling), and anti‐actin (AC‐15, Sigma‐Aldrich).
9
Protein Immunoprecipitation and Detection
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Anti-FLAG M2 magnetic beads, 3X FLAG peptide, Phalloidin-FITC, anti-Vinculin (VCL), and PP242 were obtained from Sigma-Aldrich. Anti-AU1 agarose beads were from BETHYL Lab. AU1 peptide (DTYRYI) was from COVANCE. Recombinant C-terminal fragment (amino acids 1730–2639) of Human FLNA purified from E. coli was obtained from Creative Biomart. Full-length inactive recombinant AKT1/PKB was from EMD Millipore. Anti-mTOR, Anti-phospho-AKT(Ser473), Anti-AKT, Anti-phospho-FLNA(Ser2152), Anti-phospho-S6(Ser235/236), Anti-S6, Anti-phospho-ERK (Thr202/Tyr204), and Anti-ERK were obtained from Cell Signaling. Anti-FLNA was obtained from EMD Millipore. Anti-RICTOR and anti-SIN1 were obtained from BETHYL Lab. Anti-phospho-CRKII and Anti-CRKII (Ser41) were obtained from Santa Cruz Biotechnology.
10
Protein Expression Analysis via Western Blot
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Protein extracts were prepared using high-salt lysis buffer (Hepes 50 mM , pH 7.5, NaCl 500 mM , DTT 1 mM , EDTA 1 mM , 0.1% NP-40) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). Then, 10–20 μg cell and tissues lysates were separated on 10% NuPAGE gels (Invitrogen), transferred onto a nitrocellulose membrane (Invitrogen) and incubated with the following antibodies: anti-p65BTK (BN49); anti-BTK (sc-1696) anti-hnRNPK (sc-25373) from Santa Cruz Biotechnologies; anti-ERK (#9101), anti-phospho-ERK (Thr202/Tyr204) (#4370), anti-eIF4G2 (#5169) from Cell Signaling; anti-actin (A1978), anti-vinculin (V9264), anti-phospho-hnRNPK (SAB4504229) from Sigma-Aldrich; and anti-RAS (#05-516) from Millipore. Each single blot was reprobed with anti-actin or anti-vinculin as loading control. Images were acquired using G:BOX XT4 Chemiluminescence and Fluorescence Imaging System (Syngene, Cambridge, UK) and processed with Adobe Photoshop.
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