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Figure S3. Lb2 mRNA and LB2 Protein Localize to Axons, Related to Figure 3(A and B) Lb2 and pax6 ISH in eye sections (RGC: retinal ganglion cell layer; ONH: optic nerve head; ON: optic nerve). The same sections were counterstained for axonal (neurofilament) and nuclear (DAPI) markers. The ONH, where RGC axons collect to exit the eye, is devoid of cell bodies and contains lb2 mRNA but not pax6 mRNA.(C and D) Lb2 and pax6 ISH in brain sections with a counterstained image. The tectal neuropil, where RGC axons terminate, is devoid of cell bodies and contains lb2 mRNA but not pax6 mRNA (e.g., inside the red dashed line).(E) LB2 immunostaining in brain sections shows that LB2 localizes to RGC axons in the optic tectum (Tec) (e.g., inside the red dashed line). Arrowheads indicate axons ascending to the optic tectum.(F and G) Brn3, a nuclear factor abundantly expressed in RGCs, does not localize to the tectal neuropil (e.g., inside the red dashed line) or nearby tegmental neuropil (white arrow), although it is highly expressed by a subset of neurons in the tegmentum (green arrow).(H) LB2 antibody recognizes the nucleus (red) as expected (two left panels) in fibroblast-like cells in eye explant culture. Increasing the exposure reveals localization of LB2 outside the nucleus (upper right panel). LB2MO significantly decreases the nuclear LB2 staining (arrowhead), examined at the same exposure (two middle panels). Increasing the exposure shows that the extranuclear LB2 staining (arrow) is also reduced with LB2MO, indicating that the cytoplasmic LB2 signal is specific (two right panels).(I) Western blot of stage 40 embryo head lysate shows that LB2MO inhibits LB2 translation in a dose-dependent manner.(J) Quantification of the lane intensity of LB2/α-tubulin for the indicated conditions.(K and L) Two additional independent antibodies against LB2 detect a single major band in western blot and extranuclear LB2 in axons and GCs in culture.(M) LB2MO reduces axonal LB2 detected by a different antibody confirming the specificity of this signal (∗∗p < 0.01; unpaired t test).Scale bars: 25 μm in (A)–(E), 5 μm in (F) and (J). |