Ezview red anti flag m2 affinity gel clone m2

We lysed cells in RIPA buffer and cleared of cell debris. We performed BCA protein quantification according to manufacturer guidelines (BCA Protein Assay Kit, Fisher Scientific). For immunoprecipitation of Flag-tag, we rotated 1mg lysate with Flag affinity beads (EZview™ Red ANTI-FLAG M2 Affinity Gel clone M2, Sigma-Aldrich) overnight, and then wash the unbound material with RIPA buffer. For Western-Blot analysis we loaded whole IP or equal amounts of lysate onto a 4–12% Bis-Tris gel (Life technologies), separated by SDS PAGE, and transferred to a nitrocellulose membrane for western blot analysis. We detected NT5C2 with mouse anti-NT5C2 (Sigma Aldrich, #WH0022978M2) and rabbit anti-pSer502-NT5C2 (dilution 1:1000) (Covance) antibodies and β-actin with a mouse monoclonal anti-β-actin antibody (Sigma Aldrich, #A5441).

SPRED1 plasmids were generated as previously described (Stowe et al., 2012 (link)). Additional mutants were generated by PCR-directed mutagenesis and confirmed by sequencing. Transient transfection in HEK293T cells was performed with Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, 11668019) and Opti-MEM Reduced Serum Medium, with GlutaMAX Supplement Thermo Fisher Scientific, 51985091) following manufacturer’s recommendation. Fresh media was added 16 hours after transfection and cells were lysed the follow day in lysis buffer containing 20mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, protease (Sigma Aldrich, P8340) and phosphatase inhibitors (Sigma Aldrich, P0044 and P5726). Immunoprecipitations were performed with 20 μl of EZview Red Anti-Flag M2 Affinity Gel clone M2 (Sigma-Aldrich, F2426).