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Figure 6 | BMC Cell Biology

Figure 6

From: Protein-trap version 2.1: screening for expressed proteins in mammalian cells based on their localizations.

Figure 6

The screening for a family of proteins that translocates in hypoxia. (A) Screening scheme. A single copy of EpiTag vector is inserted into the genome by replication-defective retrovirus. Following integration of the vector, cells were selected by puromycin and each puromycin-resistant colony was picked and replated to 96-well cell culture plates individually. The master plates were stored frozen and the duplicate tester plates were generated. Both tester plates were subjected to the infection by another replication-defected retrovirus expressing Cre-recombinase. One of the tester plates was cultured in normoxia and the other tester plate was cultured in hypoxia. Following incubation in each environment for 18 hrs, cells were fixed and stained with 9E10 antibody and staining patterns were analyzed using Axiovert 200 M microscope (Carl Zeiss) equipped with a motorized automated stage and fluorescence filters using Open Lab Image analysis software (Improvision). (B) Translocation of epitope-tagged EIF1 in response to hypoxia. In normoxia, epitope-tagged EIF1 is localized ubiquitously in cytoplasm in a patchy manner. In hypoxia, its localization shifts to fibrous pattern resembling actin cytoskeleton. (C) Western blot analysis of the cell lysates. The blot was probed with anti-myc antibody. The protein with the same molecular weight was detected in the lysates prepared from the cells cultured in normoxia and hypoxia conditions. The size of the protein band is in accordance with the known molecular weight of EIF1A protein.

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