Which both Notch-ICD and imp-a3 were overexpressed. The bars represent mean (6 S.E.) of 9 replicates (n = 50 in each replicate; total n for each genotype = 450). (E) The survival curves of different genotypes: ey-GAL4/+ (Black), ey-GAL4/UAS-HAimp-a3 (Grey), ey-GAL4/+; UAS-Notch-ICD/+ (Green), and ey-GAL4/UAS-HA-imp-a3; UAS-Notch-ICD/+ (Red). Note that both HA-imp-a3 and Notch-ICD Gracillin web expressing flies showed significantly reduced life span as compared to only HA-imp-a3 or Notch-ICD overexpressing flies. doi:10.1371/journal.pone.0068247.gof sgs-GAL4 driver and immunoprecipitated with anti-HA affinity beads (Sigma). For detection of Notch, we used monoclonal mouseanti-Notch (C17.9C6) antibody and for detection of HA, we used mouse anti-HA antibody at 1:1000 dilution (Sigma).Importin-a3 Mediates Nuclear Import of NotchDrosophila GeneticsAll fly stocks were maintained on standard cornmeal/yeast/ molasses/agar medium at 25uC. The imp a31(R59)/TM6C, imp a3D93/TM6B, imp a3D165/TM3, FRT82B imp a3D93/TM6B, UASp imp a1(T 2?)/CyO, UASp imp a2(T2 1-2)/TM6B, UASp imp a3(T3 2-1)/CyO stocks were obtained from Robert J. Fleming. We used the following alleles of Notch pathway components (kindly provided by S. Artavanis-Tsakonas) for genetic interaction studies: N1, Nnd-3, NAx-16172, Dl5F, SerBd-G, dx152. To generate somatic clones using the FLP/FRT system, the following stocks were used: y w hsFLP; FRT82B Ubi FP/TM6B and y w ey LP; FRT82B Ubi?GFP/TM6B which were obtained from Bloomington Drosophila Stock Center (Bloomington, IN). To generate somatic clones in salivary glands, 4? hours old embryos were subjected to a single heat shock (37uC for 45 min). For generation of the P[UAS A?imp-a3], a full-length imp-a3 cDNA with a HA tag at the aminoterminus was cloned in the pUAST vector. This construct was introduced into w1118 embryos by germline transformation according to the standard procedures. Multiple independent insertions were obtained. The UAS constructs were expressed under the MedChemExpress Hexokinase II Inhibitor II, 3-BP control of ey AL4, en AL4, sgs-GAL4 and ap-GAL4 drivers. To co-express Importin-a3 and Notch-ICD, w; UAS A?imp-a3/CyO; UAS-Notch-ICD/TM6B stock was generated by appropriate genetic crosses.Reagent (Bio-Rad) and images were captured with a Zeiss LSM510 Meta laser confocal microscope.Supporting InformationFigure S1 Overexpression of Importin-a3 specifically results in the formation of cytoplasmic aggregates of endogenous Notch protein. (A1 3) UAS-imp a1, UAS-imp a2, and UAS-imp a3 transgenes were expressed under the control of enGAL4 driver, which is expressed in posterior compartment cells of wing discs. Localization of endogenous Notch protein in anterior compartment (A1 3) and posterior compartment (B1 3) of a wing disc in which UAS-imp a1 expression was driven by en-GAL4. Similarly, localization of Notch protein in anterior compartment (C1 3) and posterior compartment (D1 3) of a wing disc in which UAS-imp a2 was overexpressed and distribution of Notch protein in anterior compartment (E1 3) and posterior compartment (F1 3) of a wing disc in which UAS-imp a3 was overexpressed. Note that there is no difference in Notch localization in anterior and posterior 23977191 compartment in case of UAS-imp a1 and UAS-imp a2 overexpression (A1 3) while presence of more number of Notch aggregates in cytoplasm of posterior compartment cells (F1 3) compare to anterior compartment cells (E1 3) in UAS-imp a3 overexpressed wing disc. Images in A3, B3, C3, D3, E3, and F3 are merges of those in A1 and A2, B.Which both Notch-ICD and imp-a3 were overexpressed. The bars represent mean (6 S.E.) of 9 replicates (n = 50 in each replicate; total n for each genotype = 450). (E) The survival curves of different genotypes: ey-GAL4/+ (Black), ey-GAL4/UAS-HAimp-a3 (Grey), ey-GAL4/+; UAS-Notch-ICD/+ (Green), and ey-GAL4/UAS-HA-imp-a3; UAS-Notch-ICD/+ (Red). Note that both HA-imp-a3 and Notch-ICD expressing flies showed significantly reduced life span as compared to only HA-imp-a3 or Notch-ICD overexpressing flies. doi:10.1371/journal.pone.0068247.gof sgs-GAL4 driver and immunoprecipitated with anti-HA affinity beads (Sigma). For detection of Notch, we used monoclonal mouseanti-Notch (C17.9C6) antibody and for detection of HA, we used mouse anti-HA antibody at 1:1000 dilution (Sigma).Importin-a3 Mediates Nuclear Import of NotchDrosophila GeneticsAll fly stocks were maintained on standard cornmeal/yeast/ molasses/agar medium at 25uC. The imp a31(R59)/TM6C, imp a3D93/TM6B, imp a3D165/TM3, FRT82B imp a3D93/TM6B, UASp imp a1(T 2?)/CyO, UASp imp a2(T2 1-2)/TM6B, UASp imp a3(T3 2-1)/CyO stocks were obtained from Robert J. Fleming. We used the following alleles of Notch pathway components (kindly provided by S. Artavanis-Tsakonas) for genetic interaction studies: N1, Nnd-3, NAx-16172, Dl5F, SerBd-G, dx152. To generate somatic clones using the FLP/FRT system, the following stocks were used: y w hsFLP; FRT82B Ubi FP/TM6B and y w ey LP; FRT82B Ubi?GFP/TM6B which were obtained from Bloomington Drosophila Stock Center (Bloomington, IN). To generate somatic clones in salivary glands, 4? hours old embryos were subjected to a single heat shock (37uC for 45 min). For generation of the P[UAS A?imp-a3], a full-length imp-a3 cDNA with a HA tag at the aminoterminus was cloned in the pUAST vector. This construct was introduced into w1118 embryos by germline transformation according to the standard procedures. Multiple independent insertions were obtained. The UAS constructs were expressed under the control of ey AL4, en AL4, sgs-GAL4 and ap-GAL4 drivers. To co-express Importin-a3 and Notch-ICD, w; UAS A?imp-a3/CyO; UAS-Notch-ICD/TM6B stock was generated by appropriate genetic crosses.Reagent (Bio-Rad) and images were captured with a Zeiss LSM510 Meta laser confocal microscope.Supporting InformationFigure S1 Overexpression of Importin-a3 specifically results in the formation of cytoplasmic aggregates of endogenous Notch protein. (A1 3) UAS-imp a1, UAS-imp a2, and UAS-imp a3 transgenes were expressed under the control of enGAL4 driver, which is expressed in posterior compartment cells of wing discs. Localization of endogenous Notch protein in anterior compartment (A1 3) and posterior compartment (B1 3) of a wing disc in which UAS-imp a1 expression was driven by en-GAL4. Similarly, localization of Notch protein in anterior compartment (C1 3) and posterior compartment (D1 3) of a wing disc in which UAS-imp a2 was overexpressed and distribution of Notch protein in anterior compartment (E1 3) and posterior compartment (F1 3) of a wing disc in which UAS-imp a3 was overexpressed. Note that there is no difference in Notch localization in anterior and posterior 23977191 compartment in case of UAS-imp a1 and UAS-imp a2 overexpression (A1 3) while presence of more number of Notch aggregates in cytoplasm of posterior compartment cells (F1 3) compare to anterior compartment cells (E1 3) in UAS-imp a3 overexpressed wing disc. Images in A3, B3, C3, D3, E3, and F3 are merges of those in A1 and A2, B.