Figure 7. Conditional protein depletion in cell culture and living Drosophila flies.

(a–d) Modulation of protein abundance in D. melanogaster. Two of the most commonly used Drosophila cell lines, as well as stably transformed living flies were used as follows. (a) K2:GFP stability in embryonic Drosophila Kc cells with 24-h recovery after transfection and after a temperature shift for 4 h from permissive (15 °C) to restrictive temperature (29 °C). CHX chase was performed with 100 μg ml⁻¹ in DMSO. (b) K2:GFP functionality detected as green fluorescence at permissive temperature. Scale bar, 2 μm. (c,d) Depletion of K2:TEV depending on a destabilizing N-terminal residue (F: phenylalanine) according to the N-end rule. Methionine (M) as control. (c) Transfection into Drosophila Schneider 2 (S2) cells with 24-h recovery after transfection and 60-h post-transfection temperature shift from 16 to 29 °C. (d) Drosophila flies were stably transformed with K2:TEV initiated by a Phe (F) residue or a M-K2:TEV control. Flies were subjected to shifts to permissive (18 °C) and restrictive temperatures (29 °C). Equal loading was further confirmed by staining of blotted membranes with probing against tubulin. CBB, Coomassie Brilliant Blue.