Fig. S1.

Effect of temperature on 4e-bp expression and food consumption. (A) Relative 4e-bp transcript levels in 4e-bp^+/+ (revertant control) and 4e-bp^−/− (deletion null) males, normalized to total input RNA. The 4e-bp–null mutant has undetectable transcript in all conditions. n = 6 vials of 25–30 flies for each temperature. (B) The 4E-BP protein is undetectable by Western blot in the null mutant. Flies were maintained at 25 °C and two independently processed 4e-bp^−/− protein samples are shown. (C and D) Cold reduces feeding. Canton-S male food intake over 24 h was determined by radioisotope-labeling of the medium (C) or CAFE assay (D). Consumption normalized to fly body mass is also shown (Right). n = number of vials or CAFE chambers, superimposed on each bar. (E) Cold results in a higher proportion of nonphosphorylated 4E-BP in a Cantonized white (wCS) line. 4E-BP, 4E-BP nonphosphorylated at Thr46; p-4E-BP, 4E-BP phosphorylated at Thr37 and/or Thr46 (see Methods for details). The two bands for phosphorylated 4E-BP resolved in these blots likely represent different phosphorylation states of the molecule. Males were used in all studies. Averages ± SEM are shown. **P < 0.01; ***P < 0.001.