For immunoblot analysis, cells were lysed with radio-immunoprecipitation buffer supplemented with protease inhibitor ‘cocktail’ (Sigma-Aldrich). Total lysates of the different T-cell subsets (40 μg) were resolved by electrophoresis through 4–12% Bis-Tris Nupage gels (Invitrogen, USA) and were transferred onto PVDF membranes (Millipore). The following primary antibodies were used: anti-ROR-α (Abcam); anti-MTNR1A (Santa Cruz), anti total and phospho-Erk1/2 (Cell Signalling), anti-total C/EBPα (Cell Signaling), anti-phospho C/EBPα (Cell Signaling), anti-Nfil3 (Santa Cruz), and anti-GADPH (Abcam). Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate as suggested by the manufacturer (Pierce).
Anti total and phospho erk1 2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti total and phospho-Erk1/2 is a laboratory product that detects both the total and phosphorylated forms of the Erk1/2 proteins. Erk1/2 (Extracellular signal-regulated kinases 1 and 2) are key components of the MAPK/ERK signaling pathway, which plays a crucial role in cellular processes such as proliferation, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti rorα, by Abcam (1 mentions)
Anti nfil3, by Santa Cruz Biotechnology (1 mentions)
Anti gadph, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Total p38 mapk, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti phospho, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
C digit blot scanner system, by LI COR (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti phospho and total p38, by Cell Signaling Technology (1 mentions)
4 protocols using anti total and phospho erk1 2
1
Immunoblot Analysis of T-Cell Subsets
2
Immunoblotting Assay for Cellular Signaling
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Anti-Sema6A and anti-Mical1 (SIGMA-Aldrich, MO, USA), anti-total and phospho-ser473-Akt, anti-total and phospho-ERK 1/2, and anti-PARP were from Cell Signaling (Milan, Italy), anti-ErbB-2, anti-ErbB-3, anti-Caspase 3, and anti-Hsp-70 (Oncogene, MA, USA), (Santa Cruz Biotechnology, CA, USA), and (Stressgen, NY, USA), anti-phospho-NDR(Thr444) was provided by Dr. Brian Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel), anti-total NDR and anti-phospho-H2B-S14, and HRP-conjugated secondary antibodies were from (Millipore, Billerica, MA, USA), and (Bio-Rad, CA, USA). For IHC, the secondary antibody was used as internal control.
3
Lung Tissue Culture Protocol for Adenocarcinoma Research
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The study was approved by our institutional Ethics Committee (Protocol number CRC 05-088 S1R2). Human lung explants were obtained from 4 patients aged between 50-60 years undergoing surgery for lung carcinoma resection. Tissues samples were collected from fresh lobectomy and transported to the laboratory in physiological Krebs’ solution (Sosroseno and Sugiatno, 2008 (link)). Human lung adenocarcinoma cell line (A549 cells) was from Abcam (# ab7910). NCS 613 (patent FR0601958) was given by J.J. Bourguignon and C. Lugnier (Faculty of pharmacy, Strasbourg). PDE antibodies used for Western blot analysis and immuno-staining were from FabGennix Inc. Anti-phospho and total ERK1/2, anti-IκBα, anti-p65-NF-κB, Anti-phospho, and total p38-MAPK were from Cell Signaling Technology. [3H]-thymidine (250 μCi) was obtained from New England Nuclear.
4
Signaling Pathways Modulation in Cells
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After preculture, the medium was replaced with fresh serum-free medium, and after 16-h culture, cells were treated with the indicated concentrations of BMP-2, TNF-α and FGF-8. Following stimulation with growth factors for 15 to 60 min, the cells were solubilized in 100 µl RIPA lysis buffer (Upstate Biotechnology, Inc., Lake Placid, NY) containing 1 mM Na 3 VO 4, 1 mM sodium fluoride, 2% sodium dodecyl sulfate, and 4% β-mercaptoethanol. The cell lysates were then subjected to SDS-PAGE/immunoblotting analysis using antiphospho-Smad1/5/8, anti-Smad1, anti-phospho-and total-ERK1/2, antiphospho-and total-P38, anti-phospho-and total-SAPK/JNK, anti-phospho-and total-NFκB-p65, anti-phospho-FGFRs (Cell Signaling Technology, Inc., Beverly, MA), anti-FGFR3 and FGFR4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-actin antibodies (Sigma-Aldrich Co. Ltd., MO). The relative integrated density of each protein band was digitized by NIH image J 1.34s or by the C-DiGit® Blot Scanner System (LI-COR Biosciences, NE).
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