Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease
J. Clin. Invest. Xiaoying Hou, et al. 109:533 doi:10.1172/JCI14099 [
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Figure 4Localization of exogenously expressed cystin in stably transfected mCCD cells. To analyze cystin localization, wild-type mCCD cells were transfected with a myc and his epitope–tagged cystin construct. Stable cell cultures were established by selection in Blasticidin. Immunofluorescence analysis was conducted in cells grown on cell culture inserts for a minimum of 3 days after confluence to allow cilial development. (
a) Schematic diagram of a principal cell with its primary apical cilium. The two focal planes used in immunofluorescence imaging are indicated. The apical focal plane was used to capture the cilium and the position of the tight junction, indicated by α-ZO-1 staining, and the nuclear focal plane identified the HOECHST-stained nuclei (blue). (
b) Immunofluorescence localization of cystin (green) as determined using the anti-his rabbit polyclonal Ab. (
c) Cystin (red) localization in the same mCCD cells as shown in
b when probed with the anti-myc mAb. (
d). Merged image of
b and
c demonstrated colocalization (yellow) of the myc and his epitope–tagged cystin. (
e) In a broad-field view, staining with the anti-his polyclonal Ab (green) indicated that cystin localized to the center of mCCD cells relative to the tight junctions stained for ZO-1 (red). (
f and
g) Broad-field and representative high-magnification views demonstrated the colocalization of the exogenously expressed cystin (red, anti-myc mAb) and endogenous polaris (green, rabbit polyclonal Ab) in cilia of mCCD cells.
