Expressed in E. coli BL21 cells at 37uC with 2 mM isopropyl-1-thio-b-Dgalactopyranoside (IPTG) induction. Bacteria were lysed in solution of Cell LyticTM express tablet (Sigma) with complete protease inhibitor (Roche). Glutathione Sepharose (GE Healthcare Bio-Sciences) beads were washed in cold phosphate buffered saline (PBS), 3 times 30 minutes each. 50 slurry was made in PBS. Intracellular domain of Notch was overexpressed in salivary glands by salivary gland specific GAL4 driver (sgs AL4) and third instar larval salivary glands were Pleuromutilin biological activity dissected and washed in PBS, then lysed in lysis buffer (50 mM Tris pH8.0, 0.1 TritonX-100, 10 Glycerol, 200 mg/ml lysozyme and 1 mM PMSF) for 3 hrs at 4uC.The supernatant was collected after centrifugation for 20 min at 12,000 rpm.Glutathione Sepharose beads alone or incubated with GST fusion proteins mixed with salivary gland lysate and rotated for 3 hrs at 4uC followed by three times washing with PBST (1X PBS, 1 Triton-X-100), 15 min each. Beads were boiled in 2X Laemmli buffer for 5 min and samples were loaded in 12 denaturing gel with Spectra multicolor broad range protein ladder used as a marker (Fermentas). Proteins were separated on nonreducing SDS-PAGE (without b-mercaptoethanol) and transferred onto PVDF membrane (Bio-Rad). Blot was probed with mouse anti-Notch C17.9C6 in 1:3000 dilution (Developmental Studies Hybridoma Bank) and secondary antibody goat anti-mouse IgGAP conjugate in 1:2000 dilution (Molecular Probes) in blocking solution (4 skimmed milk in TBST-50 mM Tris, pH 7.5, 150 mM Nacl, 0.1 Tween-20). Then after washing in TBST thrice, colour was detected by Sigma FASTTM BCIP/NBT (Sigma). Immunoprecipitation from larval salivary glands was carried out as described previously [39]. HA-Importin-a3 and Notch-ICD proteins were expressed in larval salivary glands under the controlImportin-a3 Mediates Nuclear Import of NotchFigure 5. Importin-a3 displays synergistic effect with activated Notch on signaling activity of the Notch receptor. (A1 5) Eyeantennal discs in which UAS-HA-imp-a3 was overexpressed by ey-GAL4 driver (A1 4), eye-antennal discs in which UAS-Notch-ICD was overexpressed using ey-GAL4 driver (B1 4), and eye-antennal discs from individuals in which both UAS-Notch-ICD and UAS-HA-imp-a3 were overexpressed by eyGAL4 strain (C1 4) showing Elav expression. Note that Elav staining of larval eye discs in which Notch-ICD and imp-a3 were both overexpressed showed enhanced defects in ommatidial spacing and misrotated ommatidia. Images in A3, B3, and C3 are merges of those in A1 and A2, B1 and B2, and C1 and C2, respectively. Images in A4, B4, and C4 are high magnification images of Elav expressing cells shown in A2, B2, and C2, respectively. Scale bars for A1 3, B1 3, and C1 3, 50 mm and for A4, B4, and C4, 5 mm. (A5, B5, and C5) Nail polish imprints of adult eyes of Linolenic acid methyl ester web genotypes as in A1?A4, B1 4, and C1 4, respectively. Note the co-expression of Notch-ICD and imp-a3 results in a considerable enhancement of the adult eye phenotype with more frequent fusion of ommatidia and appearance of abnormally sized ommatidia with extra bristles (arrowheads in C5). (D) Histograms show mean percentage of flies eclosed from pupae of different genotypes: ey-GAL4/+ (Black), ey-GAL4/UAS-HA-imp-a3 (Grey), ey-GAL4/+; UAS-Notch-ICD/+ (Green), and ey-GAL4/UAS-HA-imp-a3; UAS-Notch-ICD/+ (Red). Note that 58 ey-GAL4 driven Notch-ICD overexpressing pupae emerged as adult flies and this was reduced to 23 in.Expressed in E. coli BL21 cells at 37uC with 2 mM isopropyl-1-thio-b-Dgalactopyranoside (IPTG) induction. Bacteria were lysed in solution of Cell LyticTM express tablet (Sigma) with complete protease inhibitor (Roche). Glutathione Sepharose (GE Healthcare Bio-Sciences) beads were washed in cold phosphate buffered saline (PBS), 3 times 30 minutes each. 50 slurry was made in PBS. Intracellular domain of Notch was overexpressed in salivary glands by salivary gland specific GAL4 driver (sgs AL4) and third instar larval salivary glands were dissected and washed in PBS, then lysed in lysis buffer (50 mM Tris pH8.0, 0.1 TritonX-100, 10 Glycerol, 200 mg/ml lysozyme and 1 mM PMSF) for 3 hrs at 4uC.The supernatant was collected after centrifugation for 20 min at 12,000 rpm.Glutathione Sepharose beads alone or incubated with GST fusion proteins mixed with salivary gland lysate and rotated for 3 hrs at 4uC followed by three times washing with PBST (1X PBS, 1 Triton-X-100), 15 min each. Beads were boiled in 2X Laemmli buffer for 5 min and samples were loaded in 12 denaturing gel with Spectra multicolor broad range protein ladder used as a marker (Fermentas). Proteins were separated on nonreducing SDS-PAGE (without b-mercaptoethanol) and transferred onto PVDF membrane (Bio-Rad). Blot was probed with mouse anti-Notch C17.9C6 in 1:3000 dilution (Developmental Studies Hybridoma Bank) and secondary antibody goat anti-mouse IgGAP conjugate in 1:2000 dilution (Molecular Probes) in blocking solution (4 skimmed milk in TBST-50 mM Tris, pH 7.5, 150 mM Nacl, 0.1 Tween-20). Then after washing in TBST thrice, colour was detected by Sigma FASTTM BCIP/NBT (Sigma). Immunoprecipitation from larval salivary glands was carried out as described previously [39]. HA-Importin-a3 and Notch-ICD proteins were expressed in larval salivary glands under the controlImportin-a3 Mediates Nuclear Import of NotchFigure 5. Importin-a3 displays synergistic effect with activated Notch on signaling activity of the Notch receptor. (A1 5) Eyeantennal discs in which UAS-HA-imp-a3 was overexpressed by ey-GAL4 driver (A1 4), eye-antennal discs in which UAS-Notch-ICD was overexpressed using ey-GAL4 driver (B1 4), and eye-antennal discs from individuals in which both UAS-Notch-ICD and UAS-HA-imp-a3 were overexpressed by eyGAL4 strain (C1 4) showing Elav expression. Note that Elav staining of larval eye discs in which Notch-ICD and imp-a3 were both overexpressed showed enhanced defects in ommatidial spacing and misrotated ommatidia. Images in A3, B3, and C3 are merges of those in A1 and A2, B1 and B2, and C1 and C2, respectively. Images in A4, B4, and C4 are high magnification images of Elav expressing cells shown in A2, B2, and C2, respectively. Scale bars for A1 3, B1 3, and C1 3, 50 mm and for A4, B4, and C4, 5 mm. (A5, B5, and C5) Nail polish imprints of adult eyes of genotypes as in A1?A4, B1 4, and C1 4, respectively. Note the co-expression of Notch-ICD and imp-a3 results in a considerable enhancement of the adult eye phenotype with more frequent fusion of ommatidia and appearance of abnormally sized ommatidia with extra bristles (arrowheads in C5). (D) Histograms show mean percentage of flies eclosed from pupae of different genotypes: ey-GAL4/+ (Black), ey-GAL4/UAS-HA-imp-a3 (Grey), ey-GAL4/+; UAS-Notch-ICD/+ (Green), and ey-GAL4/UAS-HA-imp-a3; UAS-Notch-ICD/+ (Red). Note that 58 ey-GAL4 driven Notch-ICD overexpressing pupae emerged as adult flies and this was reduced to 23 in.
