Hrp conjugated and fluorophore conjugated secondary antibodies

Antibodies and dilutions used were mouse anti-Ptc antibody 1:50 (DSHB, Apa1; 1/50); rat anti-Ci antibody 1:50 (DSHB, 2A1; 1/50); rabbit anti-Dpp prodomain (Akiyama and Gibson, 2015 (link); 1/100); rabbit anti-GFP (Invitrogen, A-11122; 1/1000); chicken anti-GFP (Abcam Cat# ab13970; 1/2000); rabbit anti-phospho-Smad1/5 (Ser463/465) (Cell Signaling Technology, 9516; 1/100); mouse anti-HA.11 (Covance, Cat#101P; 1/1000); rabbit anti-HA (Thermo Fisher Scientific, Cat# 71-5500; 1/1000); mouse anti-β-tubulin (DSHB, E7; 1/5000); mouse anti-β-galactosidase (Promega, Z378A; 1/100); mouse anti-Myc (Santa Cruz Biotechnology, 9E10; 1/200); mouse anti-FLAG (Sigma-Aldrich, M2; 1/200); rabbit anti-FLAG (Sigma-Aldrich, Cat# F7425; 1/200); HRP-conjugated and fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch Lab and Thermo Fisher. The antibody information is also listed in the Key resources table.

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.