Ha tag

S2 cells were transfected with the indicated plasmids and lysed after 48 hours, whereas third instar larval imaginal discs were dissected and lysed directly. Lysates were immunoprecipitated, or directly subjected to SDS-PAGE and transferred to PVDF (Millipore). Membranes were immunoblotted with antibodies specific for HA tag (Invitrogen), Flag tag (Sigma), V5 tag (Invitrogen), GFP (Roche), phospho-T195-Hpo (Cell Signaling) or Tubulin (Sigma).

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM EGTA) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, and 1 μg/ml pepstatin), and a phosphatase inhibitor cocktail (Sigma). Protein concentrations were determined as above. Equal amounts of protein (25 μg) from each lysate was resolved on an 8% SDS-polyacrylamide gel (SDS-PAGE) and transferred to a PVDF membrane (BioRad). Membranes were blocked for 1h in 5% milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and incubated overnight with primary antibody at 4°C. Primary antibodies were diluted in 3% BSA-TBST. Membranes were washed in TBST four times (15min each) and incubated for 1h in appropriate horseradish peroxidase-conjugated secondary antibody (Thermo Fisher) diluted in 2% milk-TBST. Membranes were washed in TBST four times and probed with an ECL chemiluminescence kit (Pierce). Densitometry was performed using Image J software. Membranes were probed with following antibodies: rabbit polyclonal against IFIT1 (1:1000, Sigma), goat polyclonal against TGTP (1:250, Santa Cruz) and mouse monoclonal against actin (1:5000, Millipore) and HA-tag (1:1000, Thermo Scientific).