Figure 3. T194 Is an Essential Residue for Msn Activities.

(A) The plot shows the average of p-H3+ staining counts in whole midguts. The Gal4^ts drivers were crossed with the indicated UAS transgenic constructs, and the resulting flies were incubated at 29°C for 5 days, followed by gut dissection and p-H3 antibody staining. Student’s t test: **p < 0.01.

(B) Su(H)Gal4^ts and UAS transgenic flies were established and used for temperature shift, whole gut dissection, RNA isolation, and qPCR for the expression of upd3. The level of expression in the GFP control fly guts was set as 1 and the expression in the other strains was plotted as fold increase. Student’s t test: **p < 0.01.

(C–F) esgGal4^ts>GFP and UAS transgenic flies were established and used for temperature shift, gut dissection, and confocal imaging as shown. (C) esg^ts>GFP driven control midgut. (D) esg^ts>GFP driven wild-type Msn full-length protein overexpression in midgut. (E) esg^ts>GFP driven Msn full-length protein with the K61R mutation overexpression in midgut. (F) esg^ts>GFP driven Msn full-length protein with the T194A mutation overexpression in midgut. Scale bar, 20 μm.

(G) Western blots using transfected S2 cell extracts and the indicated antibodies. The cells were transfected with the indicated constructs and after 48 hr were treated with 125 nM OA or control DMSO for 1 hr and then used for extract preparation. The red arrow in lane 7 indicates a slower mobility Wts protein in the presence of co-transfected wild-type Msn.

(H) Western blots using transfected S2 cell extracts and the antibodies as indicated. A lower dose of 50 nM OA was used in these experiments. Wts^TA contained the T1077A point mutation.

(I) Western blots using transfected S2 cell extracts and the antibodies as indicated. A lower dose of 50 nM OA was used in these experiments. Wts^TA contained the T1077A point mutation. All other Wts, Msn, Hpo, and Tao were full-length constructs.

(J) Western blots using mixtures from in vitro kinase assays. GST fusion proteins were purified from bacteria expressing the constructs. The GST-WtsC contained amino acids 707–1105 spanning the C-terminal half of Wts. WtsC^TA contained the T1077A point mutation in the C-terminal fragment. Msn^KR contained the K61R point mutation, a kinase dead version. The Msn proteins were IP from transfected S2 cells, which were treated with 50 nM OA before harvesting, and then mixed with purified GST-Wts proteins in the presence of ATP for 30 min at 30°C. The mixtures were analyzed by gel separation and western blot.

The error bars are standard error of the mean.