Figure 5.

4E-BP regulates fat content. (A) w¹¹¹⁸ (“control”) or Thor² (“4E-BP null”) male flies were raised under controlled conditions and aged for 3 d on normal food or normal food supplemented with 50 μM rapamycin. Total body triglycerides and protein were measured, and the ratio of triglycerides to protein is shown. Measurements were done on batches of eight male flies in triplicate. Error bars show standard error. Average values are 5.8 μg triglycerides/μg protein (control flies, normal food), 5.6 μg triglycerides/μg protein (4E-BP-null flies, normal food), 7.5 μg triglycerides/μg protein (control flies on rapamycin), or 6.4 μg triglycerides/μg protein (4E-BP-null flies on rapamycin). (B) Control or 4E-BP-null male flies were raised under controlled conditions and aged for 3 d on normal food or normal food supplemented with 50 μM rapamycin. Flies were then starved on 0.8% agarose/PBS either supplemented with 50 μM rapamycin or not. Batches of 20 males were assayed in triplicate. Error bars show standard deviation. (C) Flies of various genotypes were grown and aged for 3 d under controlled conditions. Total body triglycerides and protein were measured for each, and the ratio of triglycerides to protein is shown. Three batches of eight males were used for each. (D) Immunoblots of S2 cells transfected to express 4E-BP or the double-alanine mutant 4E-BP(AA). Cells were treated with insulin or not as indicated to induce Tor activity. (Lower panel) Probed with antibody to 4E-BP to show relative loading. (Upper panel) Probed with antibody specific to the phosphorylated form of 4E-BP.