The antibodies used in this study were as follows: HRP-anti-hemagglutinin (12013819001) and anti-c-Myc-HRP (11814150001) (Roche Applied Science); HRP-anti-Flag (M2) (A8592) and anti-β-actin (A1978) (Sigma-Aldrich, St. Louis, MO); anti-phospho-IKKα/β (No. 2697s), anti-phospho-JNK (No.9251), anti-JNK (No. 9252) anti-phospho-ERK (No. 9101), anti-ERK (No. 9102), anti-phospho-ERK (No. 9101), anti-ERK (No. 9102), anti-phospho-p38 (No. 9211), anti-p38 (No. 9212), anti-p65/RelA (No. 6956), anti-IκBα (No. 4814) and anti-phospho-IkBα (Ser32/36) (No. 9246) (Cell Signaling Technology, Danvers, MA)); anti-IKK (Merck Millipore, Billerica, MA); Anti-LRRC25 (ab84954) (Abcam, Cambridge, U.K.); Mouse mAb HA tag DyLight 650(ab117515) (Abcam, Cambridge, U.K.). Recombinant human TNF-α was purchased from PeproTech; Lipopolysaccharides (LPS) (L4391-1 MG) and Bafilomycin A1 were purchased from Sigma-Aldrich (St. Louis, MO). Trizol was purchased from Invitrogen and Transcript First Strand cDNA Synthesis Kit from Roche.
Anti phospho erk
Manufactured by Cell Signaling Technology
Sourced in United States, Germany, China, United Kingdom
Anti-phospho-ERK is a laboratory research tool used to detect and quantify the levels of phosphorylated ERK in cellular samples. It is a specific antibody that binds to the phosphorylated form of the ERK protein, allowing researchers to measure the activation state of this important signaling molecule.
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Lab products found in correlation
Anti erk, by Cell Signaling Technology (147 mentions)
Anti phospho akt, by Cell Signaling Technology (72 mentions)
Anti phospho jnk, by Cell Signaling Technology (67 mentions)
Anti phospho p38, by Cell Signaling Technology (65 mentions)
Anti akt, by Cell Signaling Technology (60 mentions)
Anti p38, by Cell Signaling Technology (47 mentions)
Anti jnk, by Cell Signaling Technology (41 mentions)
Anti β actin, by Merck Group (28 mentions)
Anti gapdh, by Cell Signaling Technology (23 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (17 mentions)
Anti β actin, by Cell Signaling Technology (16 mentions)
Anti iκbα, by Cell Signaling Technology (14 mentions)
Anti gapdh, by Santa Cruz Biotechnology (14 mentions)
Anti bax, by Cell Signaling Technology (13 mentions)
Anti actin, by Merck Group (12 mentions)
Anti bcl xl, by Cell Signaling Technology (11 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (11 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (10 mentions)
Anti β actin, by Santa Cruz Biotechnology (10 mentions)
Anti bcl 2, by Cell Signaling Technology (10 mentions)
265 protocols using anti phospho erk
1
Antibody Characterization and Analysis
2
Western Blot Analysis of Cell Signaling
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Western blot analysis was performed as previously described [26 (link)]. Antibodies used in this study included mouse anti-HSC70 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-Shc, anti-phospho-Shc, anti-Stat5, anti-phospho-Stat5, anti- Stat3, anti-phospho-Stat3, anti-AKT, anti-phospho-AKT, anti-phospho-ERK, anti-phospho-ERK, (Cell Signaling Technology, Beverly, MA, USA). HSC70 antibodies served as loading controls.
3
Muscle Protein Expression Analysis
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The peripheral parts of muscle specimens were immediately frozen in liquid nitrogen and stored at −80°C for biochemical studies. Western blotting was performed as previously described (Fujimura and Usuki, 2015 (link), 2017 (link)). Briefly, the samples were sonicated for 5 s in tissue lysis buffer (T-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, USA) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, St Louis, USA). The samples were centrifuged (14,000 g for 1 h), and the supernatants were collected. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 μg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were then subjected to the following antibody probes: anti-MGF (Millipore, Billerica, USA); anti-IGF-I (Santa Cruz Biotechnology, CA, USA); anti-YAP1 (Novus Biologicals, Centennial, CO, USA); anti-phospho-YAP1 (Abcam, Cambridge, UK); anti-PAX7 (Cytoskeleton Incorporated, Denver, USA); anti-phospho-ERK, anti-ERK, anti-AKT, anti-phospho-AKT, anti-phospho-4EBP1, and anti-phospho-p70 S6 kinase (Cell Signaling Technologies); and anti-β-actin (Sigma-Aldrich).
4
FGF1-mediated Signaling Pathway Analysis
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Cells were treated for 45 min with either JNJ42756493 or control-treated with the corresponding concentration of JNJ427756493 diluent DMSO and for the last 15 min with or without 10 ng/mL of recombinant FGF1 (PeproTech, #100–17A, Rocky Hill, NJ, USA). Treated and control cells were then washed with PBS and were extracted using a 1.5 X Laemmli lysis buffer. Whole cell proteins (30 μg) were electrophoresed on 8–10% SDS-PAGE gels and transferred to PVDF membranes (Immobilon, Millipore, Burlington, MA, USA). Membranes were blocked with 5% BSA7030 (Sigma-Aldrich), for phosphorylated FRS2 or low-fat milk, then incubated overnight at 4°C with the following antibodies: anti-phospho-ERK (Thr202/Tyr204, #4370), anti-ERK (137F5, #4695), anti-phospho-AKT (Ser473, #4060), anti-AKT (11E7, #4685), anti-FGFR1 (D8E4, #9740), anti-FGFR4 (D3B12, #8562), anti-FGFR2 (D4L2V, #23328), anti-FGFR3 (C51F2, #4574), and anti-phosphoFRS2α (Tyr436, #3861) from Cell Signaling Technologies, anti-FRS2 (ab183492, Abcam), anti-MDM2 (Ab1-IF2, OP46, Calbiochem, Darmstadt, Germany), anti-P53 (DO-1, sc-126, Santa Cruz Biotechnology), anti-P21WAF1 (Ab-1, EA-10, Millipore), anti-βtubulin (66240–1-Ig, ProteinTech, Rosemont, IL, USA), and anti-GAPDH (60004–1-Ig, ProteinTech).
Quantification of the WB was performed using ImageJ software. Images are representative of the results obtained in three separate experiments.
Quantification of the WB was performed using ImageJ software. Images are representative of the results obtained in three separate experiments.
5
Phospho-protein Analysis of Dendritic Cells
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DCs treated with PLP-2 at 100 μg/mL or LPS at 1 μg/mL for 24 h were collected by centrifugation and washed three times with pre-cold phosphate buffer (PBS). Then total proteins of DCs were prepared using KGP950 Phosphorylated Protein Extraction kit (KeyGEN Biotech, Jiangsu Province, China). Besides, the nuclear and cytoplasmic proteins were prepared using Nuclear and Cytoplasmic Protein Extraction kit (Beyotime, China). Protein concentration was determined using BCA assay kit (Beyotime, China).
Total proteins were separated by electrophoresis using a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Membrane was blocked using Tris-buffered-saline with Tween-20 (TBST) containing 5% BSA (Solarbio) at 25 °C for 1 h, and incubated with primary rabbit anti-phospho-p38, anti-phospho-ERK or anti-phospho-JNK antibody (All from Cell Signaling Technology, USA) for 12 h at 4 °C. The membrane was then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing, China) for 1 h at 37 °C. Finally, membrane was developed with enhanced ECL kit (Beyotime, China). Chemiluminescence detection was performed on ChemiDoc XRS+ imaging system (BIO-RAD). Similarly, IκB-α in the cytoplasma extracts were also determined by Western blot analysis with anti-IκB-α antibody (Cell Signaling Technology, USA).
Total proteins were separated by electrophoresis using a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Membrane was blocked using Tris-buffered-saline with Tween-20 (TBST) containing 5% BSA (Solarbio) at 25 °C for 1 h, and incubated with primary rabbit anti-phospho-p38, anti-phospho-ERK or anti-phospho-JNK antibody (All from Cell Signaling Technology, USA) for 12 h at 4 °C. The membrane was then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing, China) for 1 h at 37 °C. Finally, membrane was developed with enhanced ECL kit (Beyotime, China). Chemiluminescence detection was performed on ChemiDoc XRS+ imaging system (BIO-RAD). Similarly, IκB-α in the cytoplasma extracts were also determined by Western blot analysis with anti-IκB-α antibody (Cell Signaling Technology, USA).
6
Western Blot Analysis of Protein Signaling
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Samples were denatured in buffer containing 60 mM Tris/pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), and 14.4 mM 2-mercaptoethanol with 0.1% bromophenol blue, and boiled for 5 minutes. SDS polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were loaded with 25 µg of total protein per lane. Prestained molecular weight markers (Bio-Rad) were used as standards. Electrophoresed samples were transferred to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ, USA). After transfer, membranes were blocked with 3% bovine serum albumin in Tris-phosphate-buffered saline (TPBS; 200 mM Tris/pH 7.0, 1.37 M NaCl, 1% Tween-20) for 1 hour at room temperature. Membranes were then incubated with anti-RET, anti-phospho-RET, anti-EGFR, anti-phospho-EGFR, anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, or anti-beta actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, washed as before, and incubated with goat anti-rabbit peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology) for 2 hours at room temperature. Membranes were then washed, and expressed proteins were detected with Pierce ECL plus western blotting substrate (Pierce, Rockford, IL, USA). Experiments were conducted at least three times independently.
7
Cordycepin Modulates AMPK and mTOR Pathways
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Cordycepin specified to be over 99.2% pure as determined by HPLC was purchased from Shanghai Winherb Medical Co. (Shanghai, China). Primary antibodies against the following proteins were obtained from Cell Signalling Technology (Danvers, MA): anti‐AMPKα (#2603P), anti‐phospho‐AMPKα (#2535), anti‐mammalian target of rapamycin (mTOR) (#2983), anti‐phospho‐ mTOR (#2971), anti‐phospho‐ERK, #4370P), anti‐ERK (#4695), anti‐acetyl‐CoA carboxylase (ACC) (#3676), anti‐phospho‐ACC (#3661), and anti‐GAPDH (#2118). Primary antibodies against gp91phox (ab129068), superoxide dismutase 1 (SOD1, ab16831), SOD2 (ab38155), and α‐actinin (ab68167) were purchased from Abcam (Cambridge, UK). The secondary antibody was purchased from LI‐COR Biosciences. Ang II (A9525) and compound C (CpC, P5499) were purchased from Sigma‐Aldrich. Proteins were measured with assay kits obtained from Pierce (Pierce, 23225).
8
Integrin-mediated Signaling Pathway Analysis
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Antibodies: Anti-phospho-ERK (#4370), -phosphop38 (#4831), phosphor-AKT (#4058), and—phospho-Smad1/5 (#9511) were from Cell Signaling (Ozyme, St Quentin en Yvelines France). Anti-β1 integrin (MB1.2) was from BD Biosciences (Le Pont de Claix, France). For IHC, β1 integrin clone 4B7R antibody (Abcam, Paris, France) was used. The anti-phosphotyrosine (PY) monoclonal antibody 4G10 used as hybridoma supernatant was produced in our laboratory. Anti-Actin (clone AC-40), anti -Type I Collagen and doxycycline were from Sigma-Aldrich (L’Isle d’Abeau, France). BMP2 was from Shenandoah Biotechnology Inc. and was used at a 200μg/mL concentration. pEl-BRE4xLuc plasmid was a gift from J. Massagué (NY, USA).
9
Antibody Reagents for Cell Signaling
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Anti-LC3B, anti-Bcl-xL, anti-Atg16L1, anti-Rac1, anti-Rho1, anti-Cdc42, anti-phospho-ERK, anti-EEA1 and anti-LAMP1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies reactive with LRP1 (11H4) were a kind gift from Dr. Strickland, University of Maryland School of Medicine, Baltimore. Fibronectin (EP5) and ubiquitin (P4D1) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); anti-Cx43, anti-ERK, anti-Bcl-2 and anti-RPTPβ antibodies were from BD Biosciences (Tokyo, Japan); anti-multi ubiquitin monoclonal antibody (FK1) was from MBL (Nagoya, Japan); anti-GAPDH antibody was from GeneTex (Irvine, CA, USA) and anti-LC3 (clone 1703) antibody was from Cosmo Bio (Tokyo, Japan). Anti-RPTPα rabbit polyclonal antibodies for immunoblotting were provided by Dr. Jan Sap; anti-α-tubulin and anti-FLAG M2 antibodies, N-acetyl-l -cysteine (NAC) and ammonium chloride were from Sigma Aldrich (St. Louis, MO, USA); ProLong Gold Antifade Reagent with DAPI and DIDS were from Invitrogen (Eugene, OR, USA); an ERK inhibitor, U0126, was from Cayman Chemical (Ann Arbor, MI, USA); Rac1 inhibitor, NSC23766 was from Wako Pure Chemical Industries (Osaka, Japan); and GSH was from Merck (Darmstadt, Germany).
10
Western Blot Analysis of Cellular Signaling
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Protein samples from cultured cells were prepared by direct lysis in 5× SDS sample buffer, followed by heating at 95 °C for 5 min. Samples were resolved on 8–12% SDS–polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5% (w/v) dried milk in Tris-buffered saline/Tween (TBST) for 1 h at room temperature, after which they were incubated with primary antibody, followed by the correct horseradish-peroxidase-conjugated secondary antibody. Blots were developed using Western Bright ECL substrate (Advansta) or Immobilon Western chemiluminescent substrate (Millipore) using a Bio-Rad Gel Doc. Antibodies used were anti-HIF-1α (catalogue no. 14179), anti-IκB-α (9242), anti-phospho-NF-κB-p65 (3033), anti-NF-κB-p65 (8242), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (catalogue no. 9212), anti-phospho-ERK (9101), anti-ERK (4695), anti-phospho-p70-S6K (catalogue no. 9205), anti-p70-S6k (9202), all purchased from Cell Signaling Technology, anti-IL-1β (R&D Systems, AF401-NA), anti-β-actin (Sigma-Aldrich, AC-74), anti-FLAG (Sigma-Aldrich, F1804), L-2HGDH (Antibody Genie, CAB7996), and D-2HGDH (Antibody Genie, CAB16213). anti-HIF-1α from Novus (catalogue no. NB100-449) was used for human blots. Secondary antibodies used were horseradish-peroxidase-conjugated anti-mouse IgG, anti-goat IgG and anti-rabbit IgG (Jackson ImmunoResearch).
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