Patient-derived tissue biopsies and harvested cells were lysed in RIPA buffer (Beyotime) for 30 min on ice. The lysate was centrifuged at 20,000 ×g for 20 min at 4 °C. The supernatant was transferred, and protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of total and nuclear proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and electro-transferred onto a nitrocellulose membrane. Membranes were incubated with antibodies to NHE1 (Santa Cruz Biotechnology), CyclinD1 (Abcam, Cambridge, UK), PCNA (Abcam), MMP-2 (Santa-Cruz), MMP-9 (Santa Cruz Biotechnology), ERK1/2 (Epitomics, Burlingame, CA), phospho-ERK1/2 (Epitomics). The secondary antibodies used for detection were HRP-conjugated anti-rabbit and anti-mouse IgG (Santa Cruz Biotechnology). Immunoreactive bands were detected by an enhanced chemiluminescence kit (EMD Millipore, Darmstadt, Germany) and results were quantitated using an image analyzer Quantity One System (Bio-Rad Laboratories, Hercules, CA). β-actin and LaminB (Santa Cruz Biotechnology) were used as loading control for total and nuclear fractions, respectively.
Erk1 2
Manufactured by Abcam
Sourced in United States, United Kingdom, China
ERK1/2 (Extracellular Signal-Regulated Kinase 1/2) is a protein that plays a key role in the Mitogen-Activated Protein Kinase (MAPK) signaling pathway. It functions as a serine/threonine protein kinase and is involved in the regulation of various cellular processes, including cell growth, proliferation, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2, by Abcam (38 mentions)
Pvdf membrane, by Merck Group (14 mentions)
β actin, by Abcam (14 mentions)
Gapdh, by Abcam (11 mentions)
E cadherin, by Abcam (8 mentions)
Bcl 2, by Abcam (8 mentions)
Bcl2 associated x (bax), by Abcam (7 mentions)
Ripa buffer, by Beyotime (6 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
Mmp 9, by Abcam (6 mentions)
P jnk, by Abcam (6 mentions)
P akt, by Abcam (5 mentions)
P akt, by Cell Signaling Technology (5 mentions)
Cyclin d1, by Abcam (4 mentions)
α sma, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
N cadherin, by Abcam (4 mentions)
P38 mapk, by Abcam (4 mentions)
Interleukin 6 (il 6), by Abcam (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
92 protocols using erk1 2
1
Protein Expression Analysis in Tissue Biopsies
2
Immunoblotting for Protein Analysis
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Cells were processed for protein extraction and western blotting as described earlier.23 (link) Immunodetection was carried out using specific antibodies against MUC4 (8G7), pY1248-HER2 (mouse monoclonal) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), pHER2, pERK1/2 (mouse monoclonal), ERK1/2, FAK and pFAK (rabbit monoclonal) (Epitomics, Burlingame, CA, USA), and β-actin (mouse monoclonal) (Sigma-Aldrich Co., St Louis, MO, USA). All secondary antibodies (Santa Cruz Biotechnology Inc.) were used at 1:2,000 dilutions. Proteins were visualized with the Super Signal West Femto Maximum sensitivity substrate kit (Thermo Fisher Scientific) and the signal was detected using an LAS-3,000 image analyzer (Fuji Photo Film Co., Tokyo, Japan).
3
Western Blot Analysis of Cellular Signaling
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Cells were lysed using RIPA buffer supplemented with protease inhibitors. 40 μg of total protein was used for Western blot analysis based on standard procedures. Protein signals were detected by ECL system. The primary antibodies were listed as follows: SIRT6 (1:1000) (Abcam, New Territories, HK); LC3 (1:2000), p62 (1:2000) (Sigma, MO, USA); AKT1 (1:1000), phos-AKT1 (1:1000) (Epitomics); ERK1/2 (1:1000), phos-ERK1/2 (1:2000), ATG5 (1:1000) (Cell Signaling Technology, Danvers, USA); beta-Tubulin (1:2000) (Abmart); Actin (1:500) (Santa Cruz Biotechnology, CA, USA).
4
Comprehensive Protein Analysis of BC Tumors
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RIPA lysis buffer containing protease inhibitors was used to lyse and BC tumor tissue samples, which were then spun for 20 minutes at 12,000 rpm at 4°C. Supernatants were then collected, with a BCA kit (Beyotime Institute of Biotechnology, Nantong, China) being used to quantify the protein contents within each sample. Samples were then boiled for 15 minutes in sodium dodecyl sulfate sample buffer, after which equal quantities of protein were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were in turn blocked for 2 hours at room temperature with 5% skim milk in TBST. Blots were then probed with rabbit polyclonal anti-RIOK1 (1:1,000, Proteintech, Wuhan, China), anti–E-cadherin (1:1,000, Proteintech), anti-vimentin (1:1,000, CST, Danvers, MA); anti–N-cadherin (1:1,000, CST); PI3K (1:1,000, CST); p-AKT (1:2,000, CST); AKT (1:1,000, CST); cyclin B1 (1:500, Proteintech); p-ERK1/2 (1:1,000, Abcam, Cambridge, UK); ERK1/2 (1:2,000, Abcam). After washing in TBST, the membran washed thrice in TBST and incubated for 2 hours with appropriate HRP-conjugated secondary antibodies (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA). Enhanced che-miluminescence (Thermo Scientific) was then employed for protein detection, with analyses being repeated in triplicate.
5
Comprehensive Molecular Profiling of CCN3
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Immunohistochemistry, Immunofluorescence, Immunoblotting and ELISA were performed as previously described [7 (link)]. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to determine the concentration of extracted protein. Levels of CCN3 in the cultured supernatants were quantified by ELISA kits (R&D Lab Inc., Minneapolis, MN, USA). MEK1/2 inhibitor U0126, NFκB inhibitor EVP4593, and Sorafenib were obtained from Selleckchem (Houston, TX, USA). Primary antibodies used for immunofluorescence, immunoblotting and/or, immunohistochemistry were as follows: CCN3, α-SMA, p-C-RAF, ERK1/2, NFκB, TIMP-2, TGF-β, and p-ERK1/2 (Abcam, Cambridge, MA, USA), RANTES and C-RAF (Cell Signaling, Beverly, MA, USA), p-MEK (Epitomics, Burlingame, CA, USA), Actin (Jackson Labs, Bar Harbor, ME, USA).
6
Western Blot Analysis of EMT Markers
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Cells were harvested and lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors for 20 min at 4°C. Equal amounts of the proteins were separated by 10% SDS‐PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against β‐actin (Beyotime Biotechnology), Pol ι (Proteintech, Rosemont, IL, USA), ETS‐1, p‐ETS‐1, E‐cadherin, Erk1/2, p‐Erk1/2 (Abcam, Cambridge, MA, USA) and N‐cadherin (Multi Sciences, Hangzhou, China). The membranes were then incubated with an HRP‐conjugated anti‐mouse or anti‐rabbit secondary antibody (Beyotime Biotechnology). The protein bands were visualized using High‐sig ECL Western Blotting Substrate (Tanon, Shanghai, China). Images were collected using the Tanon‐5200 Chemiluminescent Imaging System (Tanon). Endogenous β‐actin protein expression was detected as the internal control for each sample.
7
Protein Analysis of Mouse Renal Cortex and Podocytes
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Protein analysis was performed on mouse renal cortex tissues (Ji et al., 2017 (link)) and cultured podocytes as described previously. EGFR, p-EGFR and cleaved caspase three antibodies were purchased from Cell Signaling (Beverly, MA, United States). ERK1/2, p-ERK1/2, Bcl2, Bax and synaptopodin antibodies were purchased from Abcam (Cambridge, United Kingdom). β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).
8
Epithelial-Mesenchymal Transition Markers
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Antibodies against HNRNPA2B1, E-cadherin, vimentin, N-cadherin, MMP7, MMP9, ERK1/2 and phosphorylated proteins were purchased from Abcam (Cambridge, USA). An antibody against β-actin was purchased from Bio-world Technology (St Louis Park, MN, USA). An antibody against snail was purchased from Cell Signaling Technology (Danvers, USA). Foetal bovine serum (FBS) was purchased from Sigma Chemical (St Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium, Dulbecco’s Modified Eagle’s Medium (DMEM), and trypsin were purchased from GIBCO (Grand Island, NY, USA). ERK-inhibitor (GSK2656157) was purchased from MedChem Express (New Jersey, USA) and gemcitabine was purchased from Jkchem (Shanghai, China).
9
Molecular Signaling Pathway Analysis
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ISO was purchased from Baxter Healthcare Corporation (Deerfield, IL, USA). Primary antibodies, including rabbit anti-rat JNK (cat#: ab76125), p38 MAPK (cat#: ab170099), ERK 1/2 (cat#: ab184699), IKKβ (cat#: ab124957), NF-κB p65 (cat#: ab16502), COX2 (cat#: ab15191), iNOS (cat#: ab3523), β-actin (cat#: ab179467), lamin B (cat#: ab16048), and p-NF-κB p65 (Ser536; cat#: ab76302) were acquired from Abcam (Cambridge, UK). Rabbit anti-rat phosphorylated (p)-JNK (Thr183/Tyr185; cat#: 4668), p-p38 MAPK (Thr180/Tyr182; cat#: 4511), p-ERK1/2 (Thr202/Tyr204; cat#: 4370), and p-IKKβ (Ser180; cat#: 3671) antibodies were procured from Cell Signaling Technology, Inc. (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (cat#: 0111-01) was provided by Chemicon (Temecula, CA, USA). NF-κB activation inhibitor (NAI; cat#: S1014-67.1) and p38 MAPK activation inhibitor (SB202190; cat#: 152121-30-7) were supplied by Biomol (Plymouth Meeting, PA, USA). ROS scavenger (NAC; cat#: A7250) and iNOS activity inhibitor (1400W; cat#: W4262) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All the other reagents were obtained from Sigma unless specially stated. All suspensions were freshly prepared before use.
10
Western Blot Analysis of Mouse Heart Proteins
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Mouse heart tissue/cells were lysed for 30 min with RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Beyotime, China) on ice. The lysate was centrifuged at 12 000 rpm for 15 min at 4 °C, and the protein concentrations of the supernatant were analyzed with a BCA kit (Beyotime, China). Each sample (20 µg of protein) was separated by SDS‒PAGE gels and transferred to PVDF membranes. The membranes were immersed in 5% milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies against β‐actin (CST, 1:1000), bax (Abcam, 1:1000), caspase3 (CST, 1:1000), caspase 8 (CST, 1:1000), Cyt‐c (Proteintech, 1:1000), GPx‐4 (Abcam, 1:1000), Bcl‐2 (Affinity, 1:1000), TGF‐β (CST, 1:1000), COX‐2 (Affinity, 1:1000), VEGF A (CST, 1:1000), FAK (CST, 1:1000), ERK1/2 (CST, 1:1000), p38 (CST, 1:1000), transferrin receptor (Abcam, 1:1000), and ferroportin (Novus, 1:1000) at 4 °C overnight. Then, the membranes were washed three times with TBST, followed by incubation with secondary antibodies (1:10 000) for 1 h at room temperature. The bands were visualized by using a gel documentation system (Bio‐Rad, USA) and quantified by ImageJ software.
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