Mouse anti v5

Antibodies used for cell and embryo immunostaining were as follows: mouse anti-Dlg (1:50, Developmental Studies Hybridoma Bank), rabbit anti-GFP (1:500, Molecular Probes) and mouse anti-V5 (1:500, Sigma). For Western blotting, the following antibodies were used: rabbit anti-pan-arrestin (1:1000, Affinity Bioreagents) and mouse anti-SBP (1:200, Santa Cruz). Secondary antibodies were from Invitrogen (immunofluorescence) and LI-COR (Westerns). For immunofluorescence staining, embryos were collected on apple juice/agar plates with yeast paste at 25 °C and fixed in 4% formaldehyde in PBS/heptane, then devitellinized in methanol. Embryos were rehydrated in PBT (1 × PBS with 0.1% Tween-20, Sigma) and incubated for 2 hrs at room temperature in blocking buffer (1:1 of PBT and Roche blocking buffer, Sigma), then incubated with primary antibody diluted in blocking buffer overnight at 4 °C. Embryos were washed with PBT containing 0.1% IgG-free BSA, re-blocked with blocking buffer and incubated with fluorescent secondary antibodies (Invitrogen) diluted in blocking buffer for 1 hr at room temperature. After washes in PBT, embryos were mounted with Prolong Gold anti-fade mounting reagent with DAPI (Invitrogen), and images were acquired with Zeiss LSM 880 confocal microscope.

Antibody used in this study were guinea pig anti-Dpn, 1:1000, [42 (link)]; rat anti-Ase, 1:500, [42 (link)]; chicken anti-GFP (Abcam); rabbit anti-Brat, 1:100, [9 (link)]; mouse anti-pH3 (Cell Signaling Technology); mouse anti-V5 (Sigma-Aldrich); mouse anti-Myc (9E10); mouse anti-Flag M2 (Sigma-Aldrich); mouse anti-Lamin ADL67.10 (DSHB); mouse anti-Bruchpilot nc82, 1:10 (DSHB); guinea pig anti-Mira, 1:250; rabbit anti-Hamlet, 1:50 (home-made, [42 (link)]); rabbit anti-Dichaete, 1:1000, [65 (link)]; mouse anti-Eyeless, 1:10 (DHSB), rat anti-Grainy head, 1:1000, [14 (link)].

For the pull-down assay, GST-tagged MIF or AIF proteins immobilized glutathione Sepharose beads were incubated with 500 μg of HeLa cell lysates, washed in the lysis buffer, and eluted in the protein loading buffer. For coimmunoprecipitation, 1 mg whole-cell lysates were incubated overnight with AIF antibody (1 μg/ml) in the presence of protein A/G Sepharose (Santa Cruz Biotechnology), followed by immunoblot analysis with mouse anti-Flag antibody (Clone M1, Sigma), mouse anti-V5 (V8012, Sigma) or Goat anti-MIF (ab36146, Abcam). The proteins were separated on denaturing SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked and incubated overnight with primary antibody (50 ng/ml; mouse anti-Flag; rabbit anti-AIF; or Goat anti-MIF) at 4°C, followed by horseradish peroxidase (HRP)–conjugated donkey anti-mouse, anti-rabbit or anti-goat for 1 hour at RT. After washing, the immune complexes were detected by the SuperSignalWest Pico Chemiluminescent Substrate (Pierce).