Proteins were electrophoretically separated on a SDS-polyacrylamid gel (SDS-PAGE) and then transferred onto activated polyvinylidene difluride (PVDF) membranes (T830.1, Roth) by Western Blotting in Pierce Western Blot Transfer Buffer (35040, Thermo Fisher Scientific). Membranes were saturated in Blocking buffer (PBS, 0.1% (w/v) Tween-20, 5% (w/v) non-fat dry milk) for 1 h at room temperature and subsequently incubated with the respective antibody dilution in Blocking buffer overnight at 4°C. After washing the membranes four times for 5 min at room temperature in Washing buffer (PBS, 0.1% (w/v) Tween-20) appropriate HRP-coupled secondary antibodies (anti-mouse IgG (NA931, GE Healthcare), anti-rabbit IgG (NA934, GE Healthcare), anti-rat IgG (31470, Thermo Fisher Scientific), anti-guinea pig IgG (706-035-148, Jackson ImmunoResearch)) were applied in Blocking buffer for 2 h at room temperature. After four washing cycles for 5 min in Washing buffer Western blot signals were detected by chemiluminescence using the Immobilon Western Blot Chemiluminescence HRP substrate (WBKLS0500, Millipore).

Antibodies and antisera were used in the following dilutions: Ush (1:5,000; (Fossett et al., 2001)), GFP (1:5,000; clone [3H9] from Chromotek), FLAG (1:8,000; clone M2 from Sigma), Tubulin beta (1:8,000; clone KMX-1 from Merck Millipore), dMi-2 (1:8,000; [42]), dMTA1-like (1:10,000; [38]), Cyclin B (1:5,000; clone F2F4 from DHSB), Cyclin A (1:1,000; clone A12 from DHSB), Lamin Dm0 (1:5,000; clone ADL67.10 from DHSB), dp66 (1:10,000; [43]), dp55 (1:20,000; [44]), dMEP-1 (1:10,000; [38]), dRPD3 (1:10,000; [42]), dCHD3 (1:10,000; [45]), dPc (1:50,000; [46]), dE(z) (1:1,000; [47]), dLSD1 (1:5,000; [48]).

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