Figure 2

Dkk3 is expressed in adult mouse retina, primary Müller glia culture and a human Müller glial cell line. (A) Western blot on lysates from retina, HEK293 cells and a stable cell line expressing Dkk3 (HEK-Dkk3). A single band at 58 kDa, representing post-translationally modified Dkk3, is observed in the retina and in the Dkk3 stable cell line. Smaller bands likely represent degradation products or partially glycosylated Dkk3. (B-E) Dkk3 (green) is located in the photoreceptor inner segment region (IS), inner nuclear layer (INL) and ganglion cell layer (GCL) of adult wild type mice. There is also strong staining in the inner plexiform layer between the INL and GCL. The Müller glia marker glutamine synthetase (GS) is red and DAPI-stained nuclei are blue. The outer nuclear layer (ONL) contains the photoreceptor nuclei. Dkk3 colocalizes with GS-positive Müller glia radial fibers and cell bodies (Figure 2E,C arrows). (F-I) Confirmation of Dkk3 localization in Müller glia. Rat primary Müller glia were isolated from the retina and cultured. Dkk3 (green) is found in the same cells as the Müller glia marker glutamine synthetase (red). DAPI-stained nuclei are blue and the merged image is shown. (G) Dkk3 (green) is expressed in the perinuclear region and along processes in the human Müller glial cell line MIO-M1. Scale bar in B and F is 20 μm and in J is 100 μm. (H) Dkk3 is secreted from MIO-M1 cells. Precipitated proteins from the culture media were separated on a Western blot. The 58 kDa glycosylated Dkk3 is present in both cell lysate (L) and media (M) whereas the 38 kDa non-glycosylated form is not secreted and is only in the cell lysate fraction. β-actin was used as a control.