Northern blots were
processed using the North2South Chemiluminescent Hybridization and Detection kit, according to the manufacturer’s instructions (Pierce, Rockford, IL). For probe production see Supplemental Experimental Procedures. All procedures were carried out on 1-day-old fly heads, prior to retinal degeneration, unless otherwise specified. For western blotting, proteins were separated by electrophoresis and transferred to nitrocellulose membranes as previously described (Colley et al., 1991). For immunocytochemistry, fixation, and sucrose infiltration (or O.C.T. embedding) of fly heads was carried out as previously described (Colley et al., 1991). For each experiment, at least five individual heads were sectioned and between 50 and 100 ommatidia were observed per eye. For antibodies and microscope details see Supplemental selleck kinase inhibitor Experimental Procedures. Adult heads were fixed KU-55933 molecular weight and processed according to a modification of the methods of Baumann and Walz, as previously described (Colley et al., 1991 and Colley et al., 1995). Ultrathin sections were viewed at 80 kV on a Phillips CM120 electron microscope. For all genotypes described, at least three individual heads were sectioned and 50–100 ommatidia were observed per eye. The DNA constructs were transfected into S2 cells using the Effectene Transfection Reagent (QIAGEN Inc., Valencia, CA). Following a 7 day copper induction, cells were fixed in 2% formaldehyde in PBS for 10 min and blocked
with 1% BSA, 0.1% Triton in PBS for 30 min. For quantification of cell surface labeling, cells were observed with transmitted light. For vector identities,
DNA concentrations and additional antibody and reagent information see Supplemental Experimental Procedures. We thank Drs. W. Baehr, L. not Levin, K. Moses, A. Polans, L. Puglielli, G. Wistow, C.S. Zuker and the reviewers for valuable discussions and comments on the manuscript. The authors thank A. Gajeski, B. Larsen, A. Muller, E. Pirie, E. Solberg, and M. Sookochoff for their expert technical assistance, as well as B. Krieber and Dr. B. Ganetzky for assistance with fly stocks. Dr. J. O’Tousa provided the pGaSpeR expression vector and Dr. A. Huber provided the trp-pMT/V5 construct. We thank the following people for contributing antibodies to the study: Dr. M. Ramaswami, Dr. C. Montell, Dr. C.S. Zuker, A. Becker, M. Welsh and Dr. P. Robinson. We acknowledge Dr. D. Wassarman and R. Katzenberger for generous assistance with the S2 cell transfections. We thank R. Kalil, L. Rodenkirch, and M. Hendrickson of the W.M. Keck Laboratory for Biological Imaging and B. August and R. Massey of the UW-Med. School Electron Microscope Facility. We are grateful to C. Vang for his assistance with the computer graphics. Finally, Dr. C.S. Zuker generously provided us with the opportunity to screen the EMS-generated alleles from the Zuker Collection. This work was supported by funding from NIH EY008768 (N.J.C.), NIH AG321762 (E.E.R.