Activating transcription factor 4 (ATF4) is a key player in the integrated stress response, whose expression is subject to tight translational control. Studying its stress-provoked induction, accompanied by the general translational shutdown, is intricate because the expression of reference genes declines rapidly, and finding appropriate normalization controls is challenging. We present a protocol for human hemagglutinin-tagged ATF4 (hATF4-HA) detection and high-throughput quantification in non-stress versus stress conditions using automated and quantitative western blotting. We describe steps for seeding cells, transfecting plasmids, thapsigargin treatment, sample preparation, and target protein detection. For complete details on the use and execution of this protocol, please refer to Smirnova et al.1.

Laboratory of Regulation of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 Prague 142 20 Czech Republic

Zobrazit více v PubMed

Smirnova A.M., Hronová V., Mohammad M.P., Herrmannová A., Gunišová S., Petráčková D., Halada P., Coufal Š., Świrski M., Rendleman J., et al. Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control. Cell Rep. 2024;43 doi: 10.1016/j.celrep.2024.113976. PubMed DOI PMC

Herrmannová A., Prilepskaja T., Wagner S., Sikrova D., Zeman J., Poncova K., Valasek L.S. Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively. Nucleic Acids Res. 2020;48:1969–1984. doi: 10.1093/nar/gkz1185. PubMed DOI PMC

Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29 doi: 10.1093/nar/29.9.e45. PubMed DOI PMC