Sc 28560

Fixed oocytes (15 min in 4% PFA; Sigma Aldrich) were permeabilized in 0.1% Triton X-100 for 10 min, washed in PBS supplemented with polyvinyl alcohol (PVA, Sigma Aldrich) and incubated in primary antibodies overnight at 4 °C. The following primary antibodies diluted in PVA/PBS were used: rabbit Ankyrin B (1:150; sc-28560, Santa Cruz), goat Ankyrin B (1:150; sc-14995, Santa Cruz), mouse monoclonal anti-acetylated tubulin (1:150; T6793, Sigma Aldrich), rabbit 4E-BP1(Thr70) (1:500; cs-9455S, CST), rabbit PABP (1:150; sc-28834). Oocytes were then washed 2 × 15 min in PVA/PBS and primary antibodies were detected using relevant Alexa Fluor 488/594/647 conjugates (Invitrogen) diluted to 1:250 for 1 h at room temperature. Washed oocytes (2 × 15 min in PVA/PBS) were mounted onto slides using Vectashield Mounting Medium with DAPI (H-1500, Vector Laboratories). Inverted confocal microscope (Leica SP5) was used for sample visualization. Image quantification and assembly were performed using ImageJ and Adobe Photoshop CS3. Experiments were repeated 3x with 20–30 oocytes per group/experiment.

An exact number of cells (15–30 oocytes) was washed in PVA/PBS and frozen to −80 °C. Prepared samples were lysed in NuPAGE LDS Sample Buffer (NP0007, Thermo Fisher Scientific) and NuPAGE Sample Reducing Agent (NP0004, Thermo Fisher Scientific) and heated at 100 °C for 5 min. Proteins were separated on precast gradient 4–12% SDS–PAGE gel (Thermo Fisher Scientific) and blotted to Immobilon P membrane (Millipore) in a semidry blotting system (Biometra GmbH) at 5 mA cm⁻² for 25 min. Membranes were then blocked in 5% skimmed milk dissolved in 0.05% Tween-Tris buffer saline (TTBS), pH 7.4 for 1 h. Membranes were incubated overnight at 4 °C with the following antibodies diluted in 1% milk/TTBS: rabbit Ankyrin B (1:500; sc-28560, Santa Cruz) and rabbit GAPDH (1:30 000; G9545, Sigma Aldrich). Secondary antibodies with Peroxidase were used (711-035-152Anti-Rabbit Donkey, or 715-035-151 Anti-Mouse Donkey, both Jackson ImmunoResearch), diluted 1:7500 in 1% milk/TTBS for 1 h at room temperature. ECL (Amersham) was used for visualization of immunodetected proteins on X-ray films. The films were scanned by calibrated densitometer (GS-800, Bio-Rad Laboratories) and quantified in ImageJ. Presented images were cropped from membranes, contrast and brightness was adjusted using Adobe Photoshop CS3. Full images of segments are shown in Suppl. Fig. 5.