Ref 1

Cells were lysed in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, 5 mM NaF and 5 mM Na₃VO₄. Total cell lysates (30 µg) were resolved by 10% SDS–PAGE, transferred to nitrocellulose membranes, and incubated with 100–200 µg/mL antibodies as follows: phospho-NF-κB p65 (Ser536), total NF-κB p65, pro−/cleaved caspase-3, pro-/cleaved caspase-9, IκBα, IKKε, Ref-1 (Cell Signaling Technologies) and α-Tubulin (Sigma). For loading controls, lysates were also incubated with antibodies detecting β-Actin (Sigma). Immunoreactive bands were developed using an enhanced chemiluminescence reaction (Perkin-Elmer). Nuclear and cytoplasma protein fractions were isolated using a Nuclear Extract Kit (Active Motif) as previously described [48] (link), and the nuclear origin of these extracts was verified using anti-Ref-1 antibodies. Anti-α-Tubulin antibodies were used to exclude contamination of nuclear extracts with extranuclear proteins.

Anti-TSC2, anti-STAT3 phospho-Ser727 and Tyr705, Ref-1, Rel-A phospho-Ser365, anti-β-actin, VEGF-A, BNIP3, and anti-rpS6 phospho-Ser235/236 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-HIF-1α was purchased from BD Transduction Laboratories (Oxford, UK). Rapamycin, FL3331, and JSH23 were bought from Merck (Darmstadt, Germany), and KU-0063794 was purchased from Chemquest Ltd. (Cheshire, UK), while APX3330, APX2009, APX2011, and RN7-58 were obtained from Apexian Pharmaceuticals (Indianapolis, IN, USA). Unless stated otherwise, all other lab chemicals were obtained from Merck.