Drosophila S2R + cell culture and Western blotting

Drosophila S2R + cells were grown in Schneider's Drosophila Medium (SDM) (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS) at 25°C. Sub-confluent S2R + cells were seeded in 6-well plates and subsequently transfected using Effectene Transfection Reagent (QIAGEN). Cells were cultured for 48 hr before experiments. 10 μM (final concentration) Actinomycin D was used to block RNA synthesis, and 100 μg/ml (final concentration) cycloheximide was used to block protein synthesis. Cells were treated with the indicated drugs up to 4.5 hr before significant cell death was observed. Plasmids expressing pUbi-dGFP-Myc (0.03 ug), and pUbi-RFP-HA (0.01 ug), together with empty plasmids (to reach a total of 0.3 ug of DNA) were added in each 6-well plate during transfection. The dilution of the expression plasmid was important: we observed that too much protein expression saturates the degradation machinery and prolongs the observed half-life. S2R + cells were harvested by centrifugation and lysed in RIPA buffer. Proteins were separated on a 10% SDS-PAGE gel and analyzed by Western blotting. Quantitative Western blots were performed as previously described (Eaton et al., 2014). Images were acquired using a LI-COR Odyssey Classic imager and analyzed using NIH ImageJ.