Myrakt delta4 129

5 × 10⁴ cells were plated and transfected with control empty vector plasmid (pECE; Addgene: 26453) and constitutively active AKT plasmid (myrAkt delta4-129; Addgene: 10841). In separate experiments, the above cells were transfected with control empty vector plasmid (LZRS-Rfa; Addgene: 31601) and constitutively active MEK plasmid (L1E-1; Addgene: 21193) using lipofectamine 2000 for 48 h. Cell lysates were collected and analyzed for AKT and MEK pathway activation using western blotting.

TF3 monomer (purity: 92.4%) was isolated and purified using previously established method (15 (link)). Wortmannin and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Sigma and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Antibodies against phospho-Akt (Ser473) (p-Akt), Akt, phospho-p70S6 kinase (Thr421/Ser424) (p-p70S6K), p70S6K, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), HIF-1α, Notch-1, c-Jun N-terminal kinases (JNK), p38 and phosphor-Forkhead Box O1 (Thr24) (p-FoxO1) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against phosphor-mammalian target of rapamycin (p-mTOR) (Ser2448), mTOR were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against phosphor-4E-BP1 (Ser65/Thr70) (p-4E-BP1), c-Myc, phosphor-extracellular signal-regulated kinases 1/2 (Thr202/Tyr204) (p-ERK1/2), ERK1/2 and GAPDH were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz). Plasmids (myrAkt delta4-129, pcDNA3-Flag mTOR wt, pWZL Neo Myr Flag RPS6KB1, pET14b PHAS-I, HA-HIF1alpha-pcDNA3, 3XFlagNICD1, pMXs-hc-Myc, ODD-Luciferase-pcDNA3, VEGF promoter, cMyc promoter (TBE1/2-wt), and pCBFRE-luc) were purchased from Addgene (Cambridge, MA, USA).