3xFLAG-Morgue is expressed and functional in fly tissues.
A. The P[Elav-Gal4] or P[da-Gal4] lines were used to express Morgue-3xFLAG (P[UAS-Morgue:3xFlag]) or 3xFLAG-Morgue (P[UAS-3xFlag:Morgue]) in different independent insertion lines. Cell free lysate were analyzed by SDS/PAGE and anti-FLAG Western blots. No anti-FLAG immunoreactive band was observed in a negative control using P[elav-Gal4] and P[UAS-morgue]. B. Anti-Flag immunostaining of stage 16 embryos carrying a copy of P[elav-Gal4] and P[UAS-3XFlag:Morgue]. 3XFLAG-Morgue protein is highly expressed in the embryonic central (arrow) and peripheral (arrowhead) nervous system. C. Viability of flies expressing 3xFLAG-Morgue via P[da-Gal4]. Progeny from crosses between flies carrying different combinations of P[da-Gal4] and P[UAS-3xFlag:Morgue] or P[UAS-Morgue:3xFlag] were analyzed. Flies homozygous for P[da-Gal4] and P[UAS-Morgue:3xFlag] are completely lethal. Flies with reduced copies of P[da-Gal4] or P[UAS-Morgue:3xFlag] exhibit variable effects ranging from nearly full lethality to nearly full viability. D. Expression of Morgue-3xFLAG and 3xFLAG-Morgue proteins induce a similar cell death enhancement phenotype as that observed for wild-type Morgue when co-expressed with R/Grim. LacZ-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-LacZ]: Morgue-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-Morgue]: 3xFLAG-Morgue-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-3xFlag:Morgue]: and Morgue3xFLAG-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-Morgue:3xFlag]. Both Morgue-3x-FLAG and 3x-FLAG-Morgue proteins exhibit a similar ability to enhance R/Grim induced cell death in the eye as wild-type Morgue. All photos were taken of 1–3 day old flies.