TPC1 cells (catalog SCC147; Sigma-Aldrich) were grown in DMEM supplemented with 10% FBS and plated at 50% confluency 1 d before drug treatment. CUTC48 cells (University of Colorado’s Cell Culture Services Core) were cultured in Copland medium supplemented with 10% FBS and plated at 50% confluency 1 d before drug treatment. Protein extracts were prepared using radioimmunoprecipitation assay buffer supplemented with Halt protease/phosphatase inhibitors (PI78442; VWR). Antibodies used for Western blot analysis were anti-pERK (T202/Y204; Cell Signaling Technology, #9101), anti-ERK (ERK1/2; Cell Signaling Technology, #9102), anti-pRET (Y905; Cell Signaling Technology, #3221), anti-RET (C31B4; Cell Signaling Technology, #3223), anti-pAKT (S473; Cell Signaling Technology, #D9E), anti-AKT (C67E7; Cell Signaling Technology, #4691), anti-pSTAT3 (Cell Signaling Technology, #9131), anti-STAT3 (Cell Signaling Technology, #4904), anti-pSHP2 (Y542; Cell Signaling Technology, #3751), anti-SHP2 (D50F2; Cell Signaling Technology, #3397), anti-pFRS2a (Y196; Cell Signaling Technology, #3864), anti-FRS2 (R&D Systems, #MAB4069), anti-GAPDH (Cell Signaling Technology, #D16H11), and anti-FGFR1 (Cell Signaling Technology, #9740T). BLU6864 was provided by Blueprint Medicines.
Anti p erk t202 y204
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-ERK (T202/Y204) is a primary antibody that specifically 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 pakt s473, by Cell Signaling Technology (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti rassf1a, by Abcam (2 mentions)
Anti zeb1, by Merck Group (2 mentions)
Anti pmek s217 s221, by Cell Signaling Technology (2 mentions)
Anti dnmt3b, by Cell Signaling Technology (2 mentions)
Anti mek, by Cell Signaling Technology (2 mentions)
Anti gapdh clone d16h11xp, by Cell Signaling Technology (2 mentions)
Anti irβ ab, by Santa Cruz Biotechnology (2 mentions)
Anti pir pigfr y1162 y1163 ab, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti rabbit and anti mouse igg abs, by GE Healthcare (2 mentions)
Anti pakt t308, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
16 protocols using anti p erk t202 y204
1
Profiling Receptor Signaling in Cancer Cells
2
Hepatic Protein Expression Analysis
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Hepatic expressions of CYP2E1, peroxisome proliferator-activated receptor gamma (PPAR-γ), nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), phosphorylated map kinase kinase 4 (p-MKK4), phosphorylated c-Jun N-terminal kinases (p-JNK), p62, phosphorylated p62 (p-p62), nuclear factor erythroid 2-related factor 2 (NRF2), β-actin were determined by performing western blotting. Similar-weighted liver sections obtained from mice in each group were homogenized in lysis buffer and centrifuged. Proteins were separated by performing electrophoresis on 4–12% gradient Bis-Tris gels; transferred to Hybond ECL nitrocellulose membranes; and immunoblotted using anti-CYP2E1 (Abcam, Cambridge, UK), anti-PPAR-γ (Cell Signaling, Danvers, MA), anti-NF-κB (Cell Signaling), anti-ERK (Cell Signaling), anti-p-ERK T202/Y204 (Cell Signaling), anti-p-MKK4 S257 (Cell Signaling), anti-p-JNK T183/Y185 (Cell Signaling), anti-p62 (Cell Signaling), anti-p-p62 S349 (Cell Signaling), and anti-NRF2 (Cell Signaling) antibodies. Antibody against β-actin (Sigma-Aldrich) was used to assess equal loading.
3
Western Blot Analysis of Protein Signaling
4
Protein Expression Analysis of Cell Lysates
5
Molecular Mechanisms of AMPK Regulation
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All of the drugs, including urethane, fructose, resveratrol, compound C, and dimethyl sulfoxide (DMSO), and the mouse anti-actin, goat anti-rabbit, and goat anti-mouse IgG secondary antibodies were obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). Anti-p-AMPKT172, anti-AMPK, anti-ACCS79, anti-p-ERKT202/Y204, anti-ERK, anti-nNOSS1416, anti-nNOS, anti-eNOSS177, anti-eNOS, anti-iNOS, anti-p-RSKT359/S363, and anti-RSK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-p22-phox and anti-nitrotyrosine were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p47-phox and anti-p67-phox were purchased from Millipore (Bedford, MA, USA). Anti-Cu/Zn-SOD and anti-Mn-SOD were obtained from StressGen Biotechnologies (La Jolla, CA, USA) and Abcam (Cambridge, UK), respectively.
6
Western Blot Antibody Specifications
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The anti-LMPTP antibody (Ab) was described in 23 . The anti-GAPDH (clone D16H11XP; catalog #5174), anti-pAKT-T308 (catalog #9275), anti-pAKT-S473 (catalog #9271) and anti-pERK-T202/Y204 (catalog #9101) Abs were purchased from Cell Signaling Technology. The anti-IRβ Ab was purchased from Santa Cruz Biotechnology (clone C-19; catalog #sc-711) and the anti-pIR/pIGFR-Y1162/Y1163 Ab was purchased from Life Technologies (catalog #44-804G). Primary antibodies were used at 1:1000 dilution for Western blotting. HRP-conjugated anti-rabbit and anti-mouse IgG Abs were purchased from GE Healthcare (catalog #NA934-1ML and NA931-1ML respectively) and used at 1:3000 dilution. Unless otherwise specified, chemicals and all other reagents were purchased from Sigma-Aldrich.
7
Western Blot Analysis of p-ERK
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Cells were placed on ice and washed twice using ice-cold saline. Cells were then lysed using lysis buffer (Cell Signaling Technology, shanghai, China) containing protease inhibitor cocktail (Complete MINI, Roche, China). Cell lysates were rotated on ice for 4 cycles of 30 s rotating and 5 min rest, using a vortex. Samples were then centrifuged at 4°C at 12,500 rpm for 15 min and pellets were discarded. Protein concentration was measured using the BCA assay (Beyotime Biotechnology, shanghai, China). An equal amount of protein was loaded into the wells on an 8% bis-acrylamide gel, for SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride using the wet transfer (2.5 h at 275 mA). After overnight incubation at 4°C with 1:1000 anti-p-ERK (T202/Y204) (Cell signaling, USA), membranes were incubated with 1:10000 HRP-linked anti-Rabbit IgG (Cell Signaling Technology, shanghai, China) for 1 h. Hereafter, enhanced chemiluminescence (ECL) using ECL plus kit (Beyotime Biotechnology, shanghai, China) was used to visualize the proteins. As a loading control, 1:10000 HRP-linked GAPDH (KANGCHEN) was used.
8
Western Blot Analysis of Signaling Proteins
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Cells were lysed in the RIPA buffer (P89901, Thermo Scientific) supplemented with protease inhibitors (P1862209, Thermo Scientific). The protein concentration was detected using a BCA protein assay kit (Thermo Scientific). For western blot, 40 μg protein was separated by 8% SDS-PAGE gels and blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were incubated with the following specific antibodies: anti-pEGFR (tyr1038), anti-Akt, anti-pAkt (Ser 473), anti-pMET (Y1234/1235), anti-ERK, anti-pERK (T202/Y204), anti-PDGFRβ, anti-ATM, anti-53BP1, anti-pH2AX (S139), anti-pChk2 (T68), and anti-Chk2 (Cell Signaling Technology, Danvers, MA); and anti-EGFR, anti-Met (C-12) (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-GAPDH (Sigma-Aldrich, St. Louis, MO); then, a secondary horseradish peroxidase (HRP)-conjugate antibody was used for immunodetection. The protein bands were visualized using an ECL western blotting kit (Millipore, Bedford, MA) and densitometry analysis with AlphaEaseFC software.
9
Investigating Anserine and Carnosine Effects
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MNT-1 cells were cultured in 6-well plates for 24 h then treated with L-anserine, L-carnosine (1 and 5 μg/mL), and ACCE (1.0 × 104 and 2.0 × 104 μg/mL) for 24 or 72 h.; cells treated with PBS were used as a control. The cells were washed with PBS and lysed in cell lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.4) containing protease inhibitor and phosphatase inhibitor (Roche, Mannheim, Germany). The cell lysates were run on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore; Darmstadt, Germany). Non-specific reactivity was blocked with 5% skim milk in PBS for 1 h. The membranes were then probed with the given concentration of specific primary antibodies; 1:1000 anti-Akt, 1:1000 anti-pAkt (S473), 1:1000 anti-ERK, 1:1000 anti-pERK (T202/Y204) (Cell Signaling Technology; Danvers, MA), 1:2000 anti-tyrosinase (Invitrogen, Carlsbad, CA), and 1:10,000 anti-β-actin (Sigma Aldrich, St. Louis, MO, USA). The signals were visualized using the ECLTM Prime Western Blotting Detection System (AmershamTM, Buckinghamshire, UK) and detected using the ImageQuant LAS 4000 mini-image analyzer and ImageQuantTM TL analysis software (GE Healthcare, Buckinghamshire, UK).
10
Western Blot Protein Detection
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Proteins from whole cell extracts were detected by Western blot according to standard protocols. Primary antibodies: anti-ERK, anti-pERK (T202/Y204), anti-MEK1/2, anti-pMEK1/2 (S217/S221), anti-MGMT (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich). Secondary antibodies: anti-mouse and anti-rabbit (Li-COR Biosciences). The Odyssey® CLx Infrared Imaging System (LI-COR) was utilized for signal detection and quantification.
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