Ci 1040

The primers were synthesized and PAGE purified by IDT DNA. Fugene 6 transfection reagent was purchased from Promega while Lipofectamine RNAiMax was from Life Technologies/Invitrogen. All cell culture reagents including media, antibiotics were from GIBCO/Invitrogen. Antibodies to total Ubiquitin (clone P4D1) was from Santacruz Biotechnology, Luciferase (Millipore), pAKT, AKT (Cell Signaling), Ubiquitin, Lys63 specific (clone Apu3, Millipore), His-tag (clone H3 and C-term, Invitrogen) or Millipore (H8 clone). FLAG tag-HRP antibody (Clone M2) was purchased from Sigma Aldrich. HRP-conjugated and fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch. ON-TARGET plus siRNA to TRAF6 and scrambled control siRNA were obtained from GE Life Sciences/Dharmacon. D-Luciferin was from Xenogen Corp while GloSensor was from Promega. Erlotinib was a kind gift from Genentech. Tyrphostin AG1478 was obtained from Cayman Chemical. TNF-α antagonist (WP9QY) and IL-1R antagonist were from SantaCruz biotechnology. Recombinant IGF-1, IL-1α were from PepreoTech. CI-1040, GF109203X, NVP-AEW541 and MK2206 (Cayman Chemical), and Linsitinib (LC Laboratories). Protein A and Protein G sepharose beads were from GE HealthCare. Purified NEDD4-1 (Sigma Aldrich), Myc-tagged ubiquitin (Cat. No. U-115) UBE1 (Cat. No. E305), and UBCH5 (Cat. No. E2-616) all were from Boston Biochemicals.

The MAPK inhibitors CI‐1040 (against MEK 1/2) and JNK‐IN‐8 (against JNK 1/2/3) were obtained from Cayman Chemical and Sigma‐Aldrich, respectively. The MTH1 inhibitor TH588 was a kind offer from Prof. T. Helleday (Karolinska Institutet, Solna, Sweden). The STAT inhibitors Stattic (against STAT3) and AS1517499 (against STAT6) were purchased from Sigma‐Aldrich. Inhibitors were diluted in DMSO and stored at −20°C.