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Fig. 5 | Molecular Cancer

Fig. 5

From: A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1

Fig. 5

AKT3-174aa interacts with p-PDK1 and prevents AKT thr308 phosphorylation. a RNA sequencing was conducted in U251-circ-AKT3 IRES mut, U251-circ-AKT3, U373-circ-AKT3 IRES mut, U373-circ-AKT3 cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to find the most differentially changed signaling pathways. b Upper, U251, U373, SW1783 and Hs683 cells were overexpressed or knocked down circ-AKT3, according to their endogenous AKT3-174aa expression level. The AKT3-174aa, AKT1, AKT2, AKT3, AKT-pan, AKT-thr308, AKT-ser473, p-EGFR, PTEN level were determined. Lower, AKT1, AKT2 and AKT3 mRNA level were determined in above cells, respectively. c IP assay using AKT1, AKT2 and AKT3 antibodies were performed in indicated cells followed by immunoblot using AKT1, AKT2, AKT3, AKT-thr308, AKT-ser473 antibodies. d Total proteins from Flag-AKT3-174aa plasmid-transfected HEK293T cells were separated via SDS-PAGE. P-PDK1 was identified by LC/LC-MS in AKT3-174aa protein complex. e Mutual interaction of p-PDK1 and Flag-AKT3-174aa were determined by IP. f Left, prokaryotic purified His-tagged-PDK1, GST-tagged-AKT3-174aa and GST was subjected to GST pull down or His-IP. Right, eukaryotic purified His-tagged-PDK1, prokaryotic purified GST-tagged-AKT3-174aa and GST was subjected to GST pull down or His-IP. g Flag-tagged AKT3-174aa was transfected into U251 cells and immunofluorescence was performed using anti-Flag and anti-p-PDK1 antibody. Scale bar, 20 μm

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