Lgb 321

The anti-murine anti-VEGF monoclonal antibody B20–4.1.1 (B20) was provided by Genentech. AZD1208 and LGB-321 were acquired from AdooQ Biosciences, and sunitinib malate was obtained from Selleck Chemicals. Cycloheximide, MG-132, dimethyloxalyglycine (DMOG), and doxycycline were purchased from Sigma. Calcein AM was purchased from Invitrogen. The antibody against HIF-2α was purchased from Novus. Antibodies to HIF-1α, VHL, and VEGF were purchased from BD Transduction Laboratories. PIM1/2/3, phospho-IRS1 (S1101), HIFOH (P564), cleaved caspase 3 (CC3), and actin antibodies were purchased from Cell Signaling Technology. PIM1, CD31, and Ki67 antibodies used for immunohistochemistry were purchased from Abcam. An anti-HA monoclonal antibody was purchased from Covance, and the anti-pimonidazole mAb (Hypoxyprobe-1) was purchased from Hypoxyprobe, Inc. The GFP antibody was purchased from Sigma. All other materials and chemicals were reagent-grade.

The following antibodies were purchased from Cell Signaling Technology: anti-phospho- IRS1 (S1101 Cat # 2385), anti-phospho-S6 (S240/244, Cat # 5364), anti-phospho-TSC2 (S939 Cat # 3615), anti-TSC2 (Cat # 3990), anti-PTEN (Cat # 138G6), anti-Phospho-NF-kB p65 (S536 Cat # 93H1), anti-NF-kB p65 (Cat # D14E12), anti-phospho-4EBP1 (S65 Cat # 13443), anti-4EBP1(Cat # 9644), and anti-phospho-GSK3β (S9, Cat # 5558). Anti-IRS1 (Cat # 06–248) antibody was purchased from Merck Millipore. Anti-DDIT4 (Cat#10638–1-AP) and anti-SESN2 (Cat # 10795–1-AP) antibodies were purchased from ProteinTech Group. HRP conjugated anti-β-actin (Cat # A3854) was purchased from Sigma. HRP-linked mouse IgG (Cat # NA931V) and rabbit IgG (Cat # NAV934V) were purchased from GE Healthcare Life Sciences. IgG (Isotype control Cat # 400107), FITC conjugated anti-human CD45 (Cat # 304006), and APC conjugated anti-mouse CD45 (Cat # 103112) were purchased from BioLegend. Anti-S6 (Cat # SC74459) antibody was purchased from Santa Cruz laboratories. LGB-321 (Cat # A14420), AZD1208 (Cat # A13203), and IKK16 (# A12836) were purchased from Adooq bioscience. TNF-α (Cat # PHC3015) was purchased from Fisher scientific.
Supplementary Methods include a description of the Western Blot, RNA-Seq data analysis, GSEA analysis, RPPA, Active module analysis, Lentiviral production, the NF-κB reporter assay, and Statistics.