N Bloomington Drosophila Stock Center screen obtained. Genetic testing, as described in Figure 4 extra. In order not to bias the analysis, stick figures, genotypes were not used as an identifier for the screen. BMS-707035 Assessthe prerequisite for the development of ATM pATMT4 fly hsp70 and hsp70 with GAL4/CyO Gal4 / pATMT4 / offspring were heat for 1 h at 37 were shocked twice per day crossed up to 3 post hatch when flies were processed for TEM. For GAL4 embroidered long-term effects on the morphology of neurons GAL4/CyO hsp70 and hsp70 flies w1118 fly GAL4 / progeny were crossed were exposed to the same thermal shock. To assess the needs of ATM adults 3 d old flies hs hs ATMI and GAL4 flies were heat shocked for 1 h at 37, twice tm Possible for 12 days when flies were treated MMT.
To determine whether neurodegeneration has been gradual, were 20 30 0 4 day old female GAL4 / GMR GMRGAL4 / pATMT4 / and / GMR GAL4 pATMT4 / StringEY12388 Fly Fl managed schchen foodcontaining and turned into new bottles for 7 days. Flying for TEM were 9, 27 or 48 days treated sp Ter. Fly for FACS analysis, Elav GAL4 flies, UAS GFP w1118 ELAV GAL4, UAS GFP pATMT4 AG-490 / TM6b, Tb flies were crossed w1118 flies were crossed ELAV ELAV ATMI GFP flies to StringEY12388 / TM6b, Tb flies were crossed, fly ELAV StringEY12388 PCP / TM6b, Tb flies were crossed were crossed Elav PCP ELAV ATMI P35 fly and fly ELAV GFP flies were crossed GMR GMR P35. Crosses were maintained for 9-18 Ao td and then at 25 for 3 days prior to the third hard eyes of wandering larvae were not Tb pr produced.
The quantification of the effectiveness of mRNA knockdown ATM ATM in flies Patm, 3 days or hs hs evaluate ATMI GAL4 flies were Warmth for 1 h at 37 and total RNA was shocked isolated after using a RNeasy Mini Kit protocol of the manufacturer, h 2, 5 and 8, after heat shock. Real-time quantitative RT-PCR was performed as described and quantified. the effectiveness of the ATM protein knockdown in flies Patm GAL4 flies were crossed to hsp70 GAL4/hsp70 pATMT4 / pATMT4 flies and hsp70 GAL4/pATMT4 embryos were incubated for 2 h at 37 and heat the ankles T 1 h recover embryos were 25th Wei S eggs in Laemmli loading buffer with mercaptoethanol. Extracts of embryos were analyzed by SDS-PAGE and Western blot. Antique SIN3 body described above, and K Body polyclonal rabbit Antique ATM was raised against a recombinant protein comprising the kinase contain Dom Ne directed.
Sthesiert for electron microscopy SEM images 0-4 days old female flies were Quanta 200 environmental scanning electron microscope electronic with a gas detector secondary Re form relektronen shown. The cooling step was set to 0, head, w Keeping imaging. TEM images for the K directs the adult flies were create for 4 h at 4 in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer. Anf fixer Ngliche was then treated with 2% paraformaldehyde, 2% glutaraldehyde and 1% acid Gerbs treated in 0.1M cacodylate buffer replaced, and the samples were rotated overnight. KK Heads were washed with 0.1 M cacodylate buffer began with 2% osmium tetroxide in 0.1 M cacodylate for 2 h at room temperature, fixed washed