Anti tubulin e7

These assays were performed as described previously [43 (link), 59 (link)]. The gene-specific primers are as follows: for mouse Atp6v1c1, we used the same primers as described in [18 (link), 43 (link)]; for β-actin (expected product of 517 bp) sense 5’-CATTGAACATGGCATTGTTACC-3’ and antisense 5’-CAGCTCATAGCTCTTCTCCAGG-3’; for human ATP6V1C1 (common primers for C1-a and C1-b; expected product C1a: 219bp; C1b: 165bp) sense: 5’-ATGACTGAGTTCTGGCTTATATC-3’ and antisense: 5’-AGCTACTTTCTTAACCACTCC-3’; for human ATP6V1C2 (common primers for C2-a and C2-b; expected product C2a: 487bp; C2b: 349bp) sense: 5’-CGAATCTCTCTCAGACATGG-3’ and antisense: 5’-CTGGAAGTTCACTGGTAGTCC-3’. Anti-Atp6v1c1 (H-300) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA.). Antibodies to mTOR, phospho-T398 p70 S6 kinase, p70 S6 kinase, phospho-S473 Akt, Akt, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) and p44/42 MAPK (Erk1/2) from Cell Signaling Technology (Danvers, MA). Anti-tubulin (E7) and anti-LAMP-1 (1D4B) were from Developmental Studies Hybridoma Bank (DSHB). All assays were repeated three times.

Larvae were dissected and stained as described previously[27 (link), 67 (link)]. Following primary antibodies were used: anti-BRP (1:250)[16 (link)], anti-Tubulin (E7) (1:100) (obtained from the Developmental Studies Hybridoma Bank), anti-GFP (1:500)[41 (link)] (obtained from abcam), anti-DVGLUT (1:10,000)[19 (link)](gift from Aaron Diantonio, Washington University Medical School), anti-Liprin-α (1:500)[68 (link)](gift from Stephan Sigrist, Free University Berlin), anti-DAB [24 (link)](gift from Richard Ordway, Pennsylvania State University), and anti-dTau (1:1000)[34 (link), 35 (link)](gift from Doris Kretzschmar, Oregon Health and Science University and Daniel St. Johnston, University of Cambridge). Dylight conjugated goat anti-HRP antibody (1:1,000), Goat Cy3-, and Alexa 488 conjugated secondary antibodies against mouse, rabbit, and chicken IgG (1:1000) were obtained from Jackson ImmunoResearch, West Grove, PA.

Selected zygotes were fixed either after zona pellucida removal overnight in 2% formaldehyde or for 15 mins in absolute methanol (−20 °C) following extraction in a detergent based stabilization buffer^{78 (link)}. Primary antibodies (1 hr; 37 °C) included anti-tubulin (E-7; 1:20; Developmental Studies Hybridoma Bank, Iowa City, IA), γ-tubulin (1:500; gift of Dr. T. Stearns, Stanford University), myosin II (gift of Dr. P de Lanerolle, University of Illinois-Chicago); pericentrin (PCM; gift of Dr. S. Doxsey, University of Massachusetts Medical School); and detyrosinated anti-tubulin (glu-MTs; 1:500; Millipore, Temecula, CA). Primary antibodies were detected with appropriate fluorescein- or rhodamine-labeled secondary antibodies (1:200; Molecular Probes, Eugene, OR.). DNA was labeled with 10 µg/ml Hoechst 33345 (Sigma Chemical Corporation, St Louis, MO). Images were taken by conventional epifluorescence or laser-scanning confocal microscopy (Leica LCS, Leica Microsystems Heidelberg, Germany).