Selected cells were overlaid in 96-well plates, allowed to attach and then treated with or without TKIs (crizotinib, certinib or afatinib) for prespecified time points. Cell viability was determined by CellTiter 96 Aqueous One solution proliferation assay (Promega, Madison, WI). Experiments were performed in triplicate. Inhibitory proliferation curves and the 50% inhibitory concentration (IC50) were made using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). To highlight in an isogenic system different ALK kinase domain mutants’ effects on EML4-ALK, we recycled previously published IC50 values obtained using modified Ba/F3 cells from Friboulet et al [10 (link)]. H3122, H3122 CR1 and H3122 CR_A were lysed after exposure to TKIs or DMSO, lysates were separated, transferred to membranes and analyzed as previously described [8 (link), 16 (link)]. AKT, phospho-AKT (pS473), phospho-ERK1/2 (pT202/pY204), phospho-ALK (pY1604) and ALK were purchased from Cell Signaling Technology (Beverly, MA). EGFR was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ERK1/2 antibody was purchased from BD Transduction Laboratories (Lexington, KY). Primary antibodies were diluted 1:1000 and their recommended secondary antibodies diluted 1:10000.
Erk1 2 antibody
Manufactured by BD
The ERK1/2 antibody is a laboratory reagent used to detect the presence and measure the levels of the extracellular signal-regulated kinase 1/2 (ERK1/2) proteins in biological samples. ERK1/2 are serine/threonine protein kinases that play a central role in the regulation of cell proliferation, differentiation, and survival. The antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the activation and distribution of ERK1/2 in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Prism 6, by GraphPad (1 mentions)
Celltiter 96 aqueous one solution proliferation assay, by Promega (1 mentions)
Alectinib, by Selleck Chemicals (1 mentions)
Lorlatinib, by Selleck Chemicals (1 mentions)
Brigatinib, by Selleck Chemicals (1 mentions)
Crizotinib, by Selleck Chemicals (1 mentions)
Ceritinib, by Selleck Chemicals (1 mentions)
Phospho γh2ax, by Cell Signaling Technology (1 mentions)
Phospho chk1, by Cell Signaling Technology (1 mentions)
Phospho akt s473, by Cell Signaling Technology (1 mentions)
Phospho alk tyr1278, by Cell Signaling Technology (1 mentions)
Goat anti mouse immunoglobulin g igg, by Thermo Fisher Scientific (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
2 protocols using erk1 2 antibody
1
Evaluating TKI Inhibition on EML4-ALK Mutants
2
Immunoblotting of ALK and AKT Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were lysed on ice using hypotonic lysis buffer and immunoblotted as previously described [18] . Primary antibodies: phospho-ALK Tyr1278 (1:1,000; #3710S), pan-ALK D5F3 (1:5,000; #3633S), phospho-AKT S473 (1:5,000; #4060L), AKT (1:1,000; #9272), phospho-ERK1/2 (1:2,000; #4377S), PARP (1:1,000; #9542), RRM2 (1:1,000; #65939), phospho-Chk1 (1:1,000; #2348), phospho-γH2A.X (1:5,000; #9718), Tubulin (1:20,000; #2148) and GAPDH (1:20,000; #5147) were obtained from Cell Signaling Technology; while ERK1/2 antibody (1:5,000; #610124) was purchased from BD Transduction Laboratories. Horseradish peroxidase (HRP)-conjugated, goat anti-mouse immunoglobulin G (IgG), and goat anti-rabbit IgG (1:5,000) secondary antibodies were obtained from ThermoFisher scientific. Alectinib, brigatinib, lorlatinib, crizotinib, and ceritinib were purchased from Selleck Chemicals LLC (Houston, TX, USA).
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