α αtubulin

Immunostainings were carried out as described^{30 (link)} with the following modifications. Ovaries were dissected from 5–10 day old female flies in PBST (PBS with 0.3% Triton-100) and fixed in PBST with 4% formaldehyde for 20 min at room temperature (RT). Fixed ovaries were washed at least 3 times with PBST, permeabilized in PBS with 1% Triton X-100 for 1 h, and blocked in PBSTA (PBST with 1% bovine serum albumin) for 1 h. Samples were incubated with primary antibodies overnight at 4 °C in PBSTA. The dilutions of the primary antibodies were as follows: α-RpS5b (peptide antibody generated by Biomatik) 1:1000; α-RpS5a (peptide antibody was generated by Biomatik) 1:1000); α-αTubulin (Sigma) 1:5000; α-Orb (DSHB) 1:50; α-Dhc (DSHB) 1:50; α-cleaved Caspase 3 (Abcam), 1:200; α-ATP5A (Abcam) 1:1000; α-Aub 1:1000; α-Osk 1:500; α-Grk 1:500. Antisera were produced in the Lasko lab unless otherwise noted. Venus-tagged proteins were imaged directly under ultraviolet light.
Samples were washed and incubated in the dark with fluorescent secondary antibody (preadsorbed goat anti-rabbit Alexa Fluor555, or goat anti-mouse Alexa Fluor488, Molecular Probes, 1:500) in PBSTA for 90 min at RT. Samples were then dark washed and counterstained with DAPI, mounted in 1% DABCO (in 90% glycerol) anti-fade reagent, and examined under confocal microscopy (Zeiss LSM510).

For separating nuclear-enriched protein extracts by SDS-PAGE, equal volumes of nuclear-enriched extracts were loaded. To separate total protein extracts, equal amounts of protein were resolved by SDS-PAGE. Protein samples were subsequently blotted onto PVDF membranes. After blotting, membranes were blocked with Rotiblock (Roth) reagent and incubated with the respective primary antibody followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. HRP activity was detected using the SuperSignal West Femto Maximum Sensitivity kit (Thermo Scientific) and visualized by a LAS-4000 Mini bioimager (GE Healthcare Life Sciences). Signal intensities were quantified using Multi-Gauge software (GE Healthcare Life Sciences). Commercial antibodies used were HRP-conjugated α-HA (Roche), α-Histone H3 (Abcam), α-HSC70 (Stressgen), α-α-Tubulin (Sigma-Aldrich), α-rabbit IgG-HRP (Sigma-Aldrich) and α-mouse IgG-HRP (Sigma-Aldrich). α-SPA2 and α-COP1 antibodies were described previously in [42 (link)]. α-cry1 [67 ] and α-cry2 [68 (link)] antibodies were used to detect cry1 and cry2, respectively.