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. 2018 Apr 30;38(10):e00608-17. doi: 10.1128/MCB.00608-17

FIG 4.

FIG 4

Detection of IRS-1 ringlike structures in living cells. (A) Schematic illustration of the NLS–IRS-1–GFP/MitoRed expression vector in which human IRS-1 cDNA is cloned in frame with green fluorescent protein (GFP), an NLS, an HA tag, and MitoRed. Since the MitoDsRed2 sequence was inserted behind the T2A sequence, which encodes a viral self-cleaving peptide, this plasmid generates two independent proteins, NLS–IRS-1–GFP(HA) and MitoRed, from the same translation product. (B and C) Representative confocal images of live HeLa cells that stably express the NLS–IRS-1–GFP/MitoRed plasmid. IRS-1 ringlike structures were detected by either EGFP-associated green fluorescence (B) or Nomarski contrast (C). The red fluorescence in panel B indicates mitochondria (MitoRed). (D and E) HeLa clones expressing NLS–IRS-1–GFP/MitoRed were additionally immunolabeled with different anti-IRS-1 antibodies: anti-IRS-1(pS616) mouse monoclonal antibody (catalog no. 3193S; Cell Signaling Technology) (which recognizes the pS616 residue in the human IRS-1 molecule) (D) and anti-IRS-1(pY612) rabbit polyclonal antibody (catalog no. 44816G; Thermo Fisher Scientific) (which recognizes pY612 in human IRS-1) (E). The FarRed-conjugated secondary antibodies were utilized to avoid possible interference from MitoRed fluorescence. IR/DR, inverted repeat/direct repeat sequence; BSD, blasticidin S deaminase gene; PGK, phosphoglycerate kinase promoter; mCMV, murine cytomegalovirus early promoter; Hs IRS1, Homo sapiens IRS1 gene.