Figure 3

The 14-3-3η protein interacts directly with α-tubulin. (A-C) A single confocal section through the region of the meiotic spindle of a representative in vitro-matured egg. Sites of protein 14-3-3η and α-tubulin are indicated by the green fluorescent spots (B). The cell was counterstained with Hoechst 33342 (blue) to visualize DNA (A) and the merged image is shown (C). (D) The non-confocal, brightfield image of this cell and spindle (white box). (E) A compressed stack of seven consecutive confocal scans performed at 2 μm intervals from the bottom of the spindle to its top. A marked accumulation of the in situ PLA sites of interaction of 14-3-3η with α-tubulin was observed at the meiotic spindles in eggs and along the egg cortices about the spindles (highlighted by the white box). (F-H) A representative control egg processed simultaneously for in situ PLA by identical procedure, in absence of the primary antibodies for 14-3-3η and α-tubulin. (F) The non-confocal, brightfield image of the cell and its spindle (white box). (G) A single, confocal fluorescence scan through the spindle region of the egg cell showing no in situ PLA fluorescent reaction spot. (H) A compressed stack of all confocal scans from the bottom of the egg cell to its top, showing complete absence of in situ PLA fluorescent reaction spots. Background fluorescence was minimal (G, H). Scale bars represent 10 μm.