Cells were collected and resuspended in lysis buffer containing 40 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 17.5 mM ß-glycerophosphate, 5 mM NaF and protease inhibitors. Cell lysates were prepared by sonication and low speed centrifugation, after Micrococcal nuclease (NEB) treatment to remove RNA and DNA, and were heated at 95°C with 200 mM DTT and 1X SDS loading dye for 10 min. Samples were loaded onto 4%–20% gradient SDS-PAGE (Bio-Rad) or 16% SDS-PAGE (Invitrogen), transferred to PVDF membranes and processed for immunoblotting. Antibodies against METTL3 (15073-1-AP), m6A (MA5-33030), Actin (MAB1501), Tubulin (05–829), Vinculin (26520-1-AP), ATF4 (10835-1-AP), were from Protein Tech, Thermo-Fisher, and Millipore; PKR (3072S), P-PKR (2611S), EIF2α (9722S), P-EIF2α (3597S), were from Cell Signaling; ATF4 (ab23760) was from Abcam. All antibodies are listed in Table S6.
Atf4 ab23760
Manufactured by Abcam
ATF4 (ab23760) is a recombinant protein that corresponds to a region within the amino acids 1-351 of human Activating Transcription Factor 4 (ATF4). ATF4 is a basic leucine zipper (bZIP) transcription factor that regulates gene expression in response to various cellular stresses.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using atf4 ab23760
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METTL3-Mediated RNA Methylation Analysis
2
Protein Extraction and Immunoblotting Protocol
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Cells were collected and resuspended in lysis buffer containing 40 mM tris-HCl (pH 7.4), 6 mM MgCl2, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 17.5 mM ß-glycerophosphate, 5 mM NaF, and protease inhibitors. Cell lysates were heated at 95°C with 200 mM DTT and 1× SDS loading dye for 10 min. Samples were loaded onto 4 to 20% gradient SDS-PAGE (Bio-Rad) or 16% SDS-PAGE (Invitrogen), transferred to nitrocellulose membranes, and processed for immunoblotting. Antibodies against FXR1 (05-1529) (used for Western), actin (MAB1501), and tubulin (05-829) were from Millipore; NOLC1 (11815-1-AP), DDX21 (10528-1-AP), PolR1D (12254-1-AP), RPLP0 (11290-2-AP), RPLP2 (16805-1AP), RPL29 (15799-1-AP), RPL19 (14701-1-AP), vinculin (26520-1-AP), ATF4 (10835-1-AP), GADD34 (10449-1-AP), and FXR1 (13194-1-AP) (used for Immunoprecipitation) were from ProteinTech; PKR (3072S), P-PKR (2611S), EIF2α (9722S), P-EIF2α (3597S), and GCN2 (3302S) (used for immunoprecipitation) were from Cell Signaling Technology; c-Myc (ab32072), P-GCN2 (ab75836), GCN2 (ab134053), and ATF4 (ab23760) (used for Western blot) were from Abcam; and anti-puromycin [3RH11] (EQ0001) was from Kerafast.
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