Interdependent expression of PICK1 and ICA69 in <i>Drosophila</i>.

Abstract

<p>(A) Immunostainings for ICA69 and PICK1 in a single peptidergic neuron in the protocerebrum of adult <i>Drosophila</i> brain. HA-tagged ICA69 targeted to peptidergic cells (<i>c929-GAL4/+; UAS-ICA69-HA/+</i>) was detected in parallel with endogenous PICK1. Arrows indicate examples of PICK1/ICA69 co-localization. Scale bar, 10 µm. (B and C) <i>Drosophila</i> ICA69-HA co-immunoprecipitates PICK1 from fly head extracts. Flies were co-expressing ICA69-HA and PICK1 under the pan-neuronal elav-GAL4 driver (<i>elav-GAL4/+; UAS-ICA69-HA/UAS-PICK1A</i>). (B) Immunoprecipitation (IP) with anti-HA antibody and immunoblotting (IB) with anti-PICK1 antibody. Left lane, control without antibody; right lane, lysate. (C) Immunoprecipitation (IP) with rat anti-HA antibody (3F10) and immunoblotting (IB) with mouse anti-HA (16B12) antibody. Left lane, control without antibody; right lane, lysate. (D) Dependence of ICA69 expression on PICK1 expression in <i>Drosophila</i>. ICA69-HA immunoreactivity is shown in control brain (<i>c929-GAL4</i>, <i>PICK¹/+; UAS-ICA69-HA/+</i>) and in PICK1-null mutant brain (<i>c929-GAL4, PICK¹/PICK²; UAS-ICA69-HA/+</i>). pi, pars intercerebralis; pc, protocerebrum; ol, optic lobe. Pictures are representative of six fly brains stained in three experiments. (E) Dependence of PICK1 expression on ICA69 expression in <i>Drosophila</i>. PICK1 immunosignal in control brain without RNAi transgene (<i>ELAV-GAL4/UAS-DCR2;;TM3/+</i>) and in brain with pan-neuronal expression of ICA69 hairpin RNA (<i>ELAV-GAL4/UAS-DCR2;;UAS-ICA-RNAi/+</i>) (ICA69-RNAi). Scale bar, 100 µm. Pictures are representative of six fly brains stained in three experiments. (F) Western blotting of head extracts from control and ICA69 RNAi flies. (G) Quantification of the data in (F). Protein levels are normalized to ELAV. Data are means ± SE, <i>n</i> = 6, <i>p</i><0.05. (H and I) Quantification by RT-PCR of ICA69 mRNA (H) and PICK1 mRNA (I) levels in heads from <i>ICA69</i> knockdown (ICA69-RNAi), <i>PICK1¹</i>, and control flies. Data are means ± SE, <i>n</i> = 6 for control and ICA69-RNAi, <i>n</i> = 4 for <i>PICK1¹</i>, *<i>p</i><0.05, **<i>p</i><0.01.</p