Vials were scored approximately every 2 minutes for proboscis-extension and after a total of 30 minutes were transferred to eppendorf tubes and snap frozen in liquid nitrogen.
The supernatants of these first digestions, recovered after spinning the digestion solution at 2,000 rpm for 5 minutes, were transferred to 30 kDa Amicon centrifugal filter units (Millipore) and spun at 3,000 rpm for a minimum of 30 minutes.
After approx 30 minutes, larvae were transferred to Eppendorf tubes and centrifuged at 2500 rpm for one minute.
For RNAse treatment of squashed salivary gland chromosomes, glands were dissected in PBS and incubated in PBS with 0.1% Triton X-100 for 2 minutes, then were transferred to PBS with 0.5 mg/ml RNAseA for 8 min. For polytene chromosome squashes, heat shocks were done for 15 min at 37°C [57].
Following centrifugation at 16,000 g for 10 minutes supernatants were transferred to new tubes and 2.5 µg of anti-HIF-1α or anti-c-myc were added.
Following inhibition of mAChE by sarin for 30 minutes, crystals were transferred to 2 4 µL of OX-buffer and incubated during a time ranging from one to ten minutes.
Following centrifugation at 16,000 g for 10 minutes, supernatants were transferred to new tubes, 2.5 µg of anti-c-myc or anti-Hsp40 were added and incubated overnight at 4°C with gentle agitation.
After centrifugation at 12000 rpm for 10 minutes, supernatants were transferred to a fresh tube and stored at -80°C.
After 30 minutes, lysates were transferred into 2 mL O-ring vials (Biospec products, Techtumlab, Umeå, Sweden) containing 200 μL silica beads (Biospec products) and stored at −20°C.
After another 10 minutes, the ghosts were transferred to a water bath at 37°C for 25 minutes to complete the resealing process.
