Newly eclosed males have been theshfted to 31 C or 29 C for two weeks prior to dssecton.Stat92E RNA and Zfh1 RNA have been nduced c587 GAL4,UAS stat92ERNA,tub GAL80ts or c587 GAL4,UAS zfh1 RNA,tub GAL80ts males by shftng newly eclosed males rased at 18 C to 31 C for 1 week before dssecton.For stuhybrdzatoand qPCR experments, newly eclosedhs upd orhs kemales wereheat shocked for 45 mnutes at 37 C and theallowed to recover for 1hour at 25 C.Mosac analyss kemutant alleles ken1, ken02970, and kenk11035 were recombned onto FRT42B chromosomes and crossed to FRT42B Ub GFP,nls,hsFLfles.The FLmedated mtotc recombnatotechnque was employed to produce negatvely marked kehomozygous mutant GSC and or CySC clones.Newly eclosed males on the genotype,PFRT G13 ke PFRT G13 PGFP,nls,MKRS, and,PFRT G13 PFRT G13 PGFP,nls,MKRS, wereheat shocked three tmes for 30 mnutes at 37 C, thedssected 2, 6, 10, and 14 days just after clone nducton.Negatvely marked GSC clones have been dentfed by ther absence of GFand the somatc markers ZFH1 or Traffc jam and by ther postoadjacent to thehub.
Negatvely marked CySC clones had been dentfed by ther absence of GFP, presence of ZFH1 or Tj, and postowth2 cell dameters from thehub.Statstcal analyss opercentage testes wth clones was performed usng the Fsher Exact or Ch Squared tests.stuhybrdzatoTo create probes for stuhybrdzaton, cDNAs for keand Ptp61F have been PCR amplfed wth prmers that contaned restrctoenzyme stes Xba and EcoR kinase inhibitor JAK Inhibitor on the 5 ends to allow for subsequent clonng.PCR amplfed products were dgested wth Xba and EcoR, and thelgated nto the pBluescrpt KS vector.Dgoxgenlabeled ant sense RNA probes were transcrbed vtro usng T3 RNA polymerase accordng to your suppliers nstructons from plasmd templates lnearzed wth Xba.Control sense probes had been transcrbed wth T7 RNA polymerase selleck chemicals from plasmds lnearzed wth EcoR.stuhybrdzatons have been carried out as descrbed and vsualzed wth aOlympus BX51 mcroscope.mmunostanng Testes were dssected from newly eclosed fles and were fxed and mmunostaned as prevously descrbed.
To vsualze keexpressothe keenhancer tralnes, tyramde sgnal amplfcatowas used to ncrease senstvty within the ant galactosdase stanng accordng for the suppliers nstructons.Antbodes utilised had been rabbt ant Vasa, rabbt ant GFP, mouse ant GAL, affnty purfed rabbt ant

Stat92E, gunea pg ant ZFH1, mouse monoclonal antbody 1B1, rabbt ant phosphohstoneh3.Alexa 488 and Alexa 568 conjugated secondary antbodes have been employed.DNA was counterstaned wth 4,6 damdno two phenylndole.Confocal mages were acqured wth a Zess LSM 5 Pascal mcroscope and fgures were assembled wth Adobe PhotoshoCS3 and Adobe lustrator CS3.