Cells were rinsed in lysis buffer (125 mM Tris-HCl, 750 mM NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mM MgCl2, 5 mM EDTA, 25 mM NaF, 1 mM NaVO4, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, pH 7.5), sonicated and centrifuged at 13,000 x g for 10 min at 4°C. 20 μg cell lysates were subjected to Western blotting and probed with the following antibodies: phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2 (Millipore); ERK1/2 (Millipore); HIF-1α (BD Bioscience, San Jose, CA); FPPS (Abcam, Cambridge, UK); Pgp/ABCB1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); MRP1/ABCC1 (Abcam); MRP2/ABCC2 (Abcam); MRP3/ABCC3 (Santa Cruz Biotechnology Inc.); MRP4/ABCC4 (Abcam); MRP5/ABCC5 (Santa Cruz Biotechnology Inc.); BCRP/ABCG2 (Santa Cruz Biotechnology Inc.); β-tubulin (Santa Cruz Biotechnology Inc.), followed by the secondary peroxidase-conjugated antibodies (Bio-Rad Laboratories). Proteins were detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA). To assess HIF-1α phosphorylation, the whole cell lysate was immunoprecipitated with a polyclonal anti-HIF-1α antibody (Santa Cruz Biotechnology Inc.), then resolved by SDS-PAGE and probed with a biotin-conjugated anti-phosphoserine antibody (Sigma-Aldrich), followed by polymeric streptavidin-horseradish peroxidase-conjugates (Sigma-Aldrich).
Erk1 2
Manufactured by Merck Group
Sourced in United States
ERK1/2 is a laboratory equipment product that functions as a kinase, a type of enzyme that catalyzes the transfer of phosphate groups to specific substrates. It is a key component in the mitogen-activated protein kinase (MAPK) signaling pathway, which is involved in various cellular processes such as cell growth, proliferation, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Phospho erk1 2, by Cell Signaling Technology (7 mentions)
β actin, by Merck Group (7 mentions)
P erk1 2, by Cell Signaling Technology (6 mentions)
Gapdh, by Merck Group (6 mentions)
P erk1 2, by Merck Group (6 mentions)
Mek1 2, by Cell Signaling Technology (5 mentions)
Bcl 2, by Merck Group (4 mentions)
Bcl2 associated x (bax), by Merck Group (3 mentions)
β tubulin, by Santa Cruz Biotechnology (2 mentions)
Sp600125, by Merck Group (2 mentions)
Sb203580, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Hmgb1, by Merck Group (2 mentions)
Phospho akt, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Alexa fluor 680 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse hrp, by Jackson ImmunoResearch (2 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (2 mentions)
49 protocols using erk1 2
1
Protein Expression Analysis in Cell Lysates
2
Signaling Pathway Modulators in Cell Culture
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Cell culture reagents were purchased from the Gibco Co. (Grand Island, NY, USA). Dimethyl sulphoxide, PD98059 [an extracellular regulated kinase (ERK1/2) inhibitor], SB203580 (a p38 kinase inhibitor), SP600125 [a Jun kinase (JNK) inhibitor], SB431542 (a type I TGF-β receptor inhibitor), LY294002 [a phosphoinositide-3 kinase (PI3K) inhibitor] and AG490 [a Janus kinase (JAK2) inhibitor] were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA). S100B was purchased from the Abcam Co. (Cambridge, UK).
The antibodies used were: S100B, collagen IV (col4α1), cyclooxygenase 2 (COX2), pan-cadherin (Abcam); Steap4 (Novus Biologicals, Littleton, CO, USA), fibronectin and RAGE (Chemicon, Temecula, CA, USA), ERK1/2, p-ERK1/2 (Thr202/Tyr204), p38 kinase, p-p38 kinase (Thr180/Try182), Akt, p-Akt (Thr308), Signal transducer and activator of transcription 3 (Stat3), p-Stat3 (Tyr705), Smad2/3, p-Smad2/3 (Ser433/435) (Cell Signaling Technology Inc., Danvers, MA, USA), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-tubulin (Thermo Scientific, Fremont, CA, USA).
The antibodies used were: S100B, collagen IV (col4α1), cyclooxygenase 2 (COX2), pan-cadherin (Abcam); Steap4 (Novus Biologicals, Littleton, CO, USA), fibronectin and RAGE (Chemicon, Temecula, CA, USA), ERK1/2, p-ERK1/2 (Thr202/Tyr204), p38 kinase, p-p38 kinase (Thr180/Try182), Akt, p-Akt (Thr308), Signal transducer and activator of transcription 3 (Stat3), p-Stat3 (Tyr705), Smad2/3, p-Smad2/3 (Ser433/435) (Cell Signaling Technology Inc., Danvers, MA, USA), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-tubulin (Thermo Scientific, Fremont, CA, USA).
3
Western Blot Analysis of Signaling Proteins
4
Investigating Luteolin and Selumetinib Effects
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HCT-116/HT-29 cells (2 × 106 cells) were seeded in a 100-mm dish. After treatment with luteolin and/or selumetinib for 48 h, cells were collected in PBS and lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Beijing, China) on ice for 30 min, and lysates were harvested by centrifugal separation. Protein concentration was determined using the BCA Protein Assay Kit (Takara). Identical amounts of protein were separated using 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were clogged with 5% nonfat dry milk for 1 h at room temperature. The primary antibodies BCL-2, BAX, cleaved caspase-3, cyclin B1, p-CHK1, CDC2, XRCC1, LIG1, HMGB1, ERCC3, p-MEK1, MEK1, p-ERK1/2, ERK1/2, and GAPDH (1:1000 dilution, Sigma-Aldrich) were added after overnight incubation at 4°C. After three washes with TBST, the membranes were incubated with anti-mouse horseradish peroxidase (HRP)-linked secondary antibodies (Beyotime Institute of Biotechnology, Beijing, China) for 1 h. The intensity of protein expression was measured using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA) [31 (link),32 ].
5
Western Blot Analysis of Protein Samples
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Protein samples were denatured by boiling in SDS sample buffer, and then electrophoresed in 7.5%, 10%, or 12% polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane, then immunoblotted with appropriate primary antibodies [Dicer1 (Abcam, 1:500), Ago2 (Abcam, 1:500), Tau (Abcam, 1:500), α-tubulin (Abcam, 1:5000), GFP (Covance, 1:300), gERK (ERK 1/2, Sigma, 1:20000)] followed by HRP-conjugated secondary antibodies (1:10000, Jackson Laboratories) and visualized using myECL Imager (Thermo), according to the manufacturer's instructions. Quantification was performed using Image J software.
6
Protein Extraction and Western Blot Analysis
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Lysates were isolated from the whole tissue homogenates or gastric cancer cells using a Total Protein Extraction kit (Millipore, Billerica, MA, USA) and were cytoplasmic and nuclear protein extracted using a Cytoplasmic and Nuclear Protein Extraction kit (Promega, Madison, WI, USA) and were subjected to western blot analysis. Antibodies against HMGB1 (1:8,000; Sigma), LC3B, ERK1/2, phospho-ERK1/2, p38, p38, AKT, phospho-AKT, JNK, phospho-JNK (1:1,000; Cell Signaling Technology, Danvers, MA, USA), RAGE (1:200; R&D Systems), β-tubulin, albumin and lamin B (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to develop immunoreactive signals. Densitometry was performed using AlphaImager 2200 system and Quantity software.
7
Comprehensive Cell Lysis and Immunoblotting
8
Western Blot Analysis of RNF135 and ERK
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Cell lysates were prepared using radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology). The concentration of protein lysate was determined with Pierce Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 20 µg protein were loaded per lane and separated via SDS-PAGE on an 8% gel. The separated proteins were then transferred to polyvinylidene difluoride membranes and subsequently blocked with 5% non-fat milk at room temperature for 1 h. Then membranes were incubated with primary antibodies anti-RNF135 (cat. no. ab28636; 1:1,000; Abcam), anti-GAPDH (cat. no. G8795; 1:8,000; Sigma-Aldrich; Merck KGaA), ERK1/2 (cat. no. 4695; 1:1,000; Cell Signaling Technology, Inc.) and phosphorylated (p-) ERK1/2 (cat. no. 9101; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Following primary incubation, membranes were incubated with horseradish peroxidase-labeled secondary mouse antibody (cat. no. AP308P; 1:10,000; Sigma-Aldrich; Merck KGaA) and rabbit antibody (cat. no. SAB3700852; 1:10,000; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Subsequently, the bands were visualized using enhanced chemiluminescence western blotting detection reagents (GE Healthcare Life Sciences). Images were captured and analyzed using ImageQuant TL 7.0 (GE Healthcare Life Sciences). GAPDH served as a loading control for protein quantification.
9
Protein Extraction and Western Blot Analysis
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Cell protein extracts were obtained using the CelLytic Mammalian Cell Lysis/Extraction Reagent (Sigma-Aldrich) with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Thirty micrograms of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (GE HealthCare, Freiburg, Germany). The membranes were blocked in 5% non-fat dry milk in Tris-buffered saline containing 10% Tween 20. Antibodies against SET (E15; #sc-5655; Santa Cruz), ERK1/2 (M 5670; Sigma-Aldrich), phospho-p44/42 MAPK ERK1/2 (pERK1/2; #4377; Cell Signaling, Rockford, IL, USA), PP2Ac (PP2A catalytic subunit; #2038; Cell Signaling), p53 (7 F5; #2527; Cell Signaling), phospho-p53 (p-p53Ser15; #9286; Cell Signaling), p21WAF1/Cip1 (12D1; #2974; Cell Signaling), β-actin and tubulin were used. The reactions were developed using the chemiluminescent ECL Western blotting system (GE HealthCare). Densitometric analysis was performed using the ImageJ 6.4 software [43 (link)], and bands were normalized to constitutive proteins. The values are presented as the shSET/shControl ratio. For phosphorylation analysis, the phosphorylated/total protein ratio was calculated, and representative values are presented.
10
Antibody Validation for Signaling Pathways
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Antibodies used were HA (SC-805, Santa Cruz, Dallas, TX), HA-7-HRP (H6533, Sigma-Aldrich), MEK1/2 (#9126, Cell Signaling, Danvers, MA), phospho-MEK1/2 (#2338, Cell Signaling), ERK1/2 (M5670, Sigma-Aldrich), phospho- ERK1/2 (#4370, Cell Signaling), STAT5 (610191, BD Biosciences, Franklin Lakes, NJ), phospho-STAT5A/B (05–886R, Merck Millipore), phospho-p70 S6 kinase (#9234, Cell Signaling), p70 S6 kinase (SC-230, Santa Cruz), RIPK3 (#12107, Cell Signaling), HSP90 (610418, BD Transduction Laboratories), actin (AAN01-A, Cytoskeleton, Denver, CO), and tubulin (ab7291, Abcam, Cambridge, UK). The secondary antibodies used were goat anti-mouse HRP (115–035-003, Jackson ImmunoResearch, West Grove, PA), goat anti-rabbit HRP (111–035-003, Jackson ImmunoResearch), and Alexa Fluor 680 goat anti-mouse (A-21057, Molecular probes, Grand Island, NY).
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