Ic261

293T (ATCC CRL-3216) cells and C2C12 (ATCC CRL-1772) were cultured in DMEM supplemented with 10% fetal bovine serum (GenDEPOT). For immunoprecipitations, 2 × 10⁶ cells were plated into 60 mm dishes 1 day before transfection, and iMFectin (GenDEPOT) was used to transfect DNA according to the manufacturer’s protocol. M2 agarose bead (Sigma) was used to pull down flag tagged proteins. TCAP degradation assays were performed by transfecting pCMV10-3XFlag-TCAP with or without Fbxl3 and Fbxl21 expression constructs into 2 × 10⁵ 293T cells in a 12-well plate. Thirty-six hours after transfection 100 μg/ml cycloheximide (CHX) was added and cells collected at the indicated times. The incubation time length was chosen to avoid significant cytotoxicity while still sufficient to distinguish effects. Half-life was determined by using nonlinear, one-phase decay analysis (GraphPad Prism). siRNA for hFbxl21 was purchased from Dharmacon and transfection was performed by using Lipofectamine 2000 (Invitrogen). CHIR99021, Ro3306, NU7441, Sotrastaurin, and IC261 were purchased from Selleckchem.

HEK293T, MCF7 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Macgene). HCT116 cells were cultured in McCoy’s 5A medium (Macgene). hTERT-RPE1 cells were cultured in DMEM/F12 medium (Gibco). Penicillin, streptomycin and 10% foetal bovine serum were added to all culture media unless otherwise noted. D4476 was purchased from Tocris Bioscience. IC261 was purchased from Selleckchem. Rabbit anti-Dvl2 and rabbit anti-PP2A/C antibodies were purchased from Cell Signaling Technology. Mouse anti-Dvl2 (10B5) and mouse anti-Myc antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-Dvl2 (phospho S143) and rabbit anti-γ-tubulin antibodies were purchased from Abcam. Mouse anti-PP5 antibody (for Western blotting) was purchased from BD Transduction Laboratories. Rabbit anti-PP5 antibody (for immunofluorescence) was purchased from Bethyl. Mouse anti-FLAG, mouse anti-acetylated tubulin and mouse anti-γ-tubulin antibodies were purchased from Sigma-Aldrich. Mouse anti-β-actin antibody was purchased from Sungene.