Fig. 3: Expression of and interaction of Occludin with Shp2.

TRAP isolated mRNA from input, RPE, rod, cone, and Müller cells were subjected to qRT-PCR with Occludin primers, and the data was normalized to the Rpl37 gene (A). Data are mean ± SEM (n = 3). Retina and RPE proteins from C57Bl6 mice were immunoblotted with Occludin, rhodopsin, RPE65, and actin antibodies (B). Rod outer segments (ROS) and rod-enriched inner segments (RIS) were immunoblotted with Occludin and rhodopsin (C). Retinal proteins from Shp2^flox/flox and ^rodShp2^−/− mice were immunoblotted with Occludin and actin antibodies (D). Densitometric analysis of OCLN was normalized to actin (E). Data are mean ± SEM (n = 3). RPE flat mounts from 20-week old Shp2^flox/flox (F) and ^rodShp2^−/− (G) mice were stained with Phalloidin and Occludin. Interaction between Shp2 and Occludin in a heterologous system. HEK-293T cells were transfected with WT-HA-Shp2, and mutant construct of Shp2, HA-Shp2(D425A), and HA-Shp2 (D425A/C459S) and 48 h later, proteins were immunoblotted with HA, Shp2 and actin antibodies (H). HEK-293T cells were transfected alone with Flag-OCLN or co-transfected with WT-HA-Shp2, and mutant construct of Shp2, HA-Shp2(D425A), and HA-Shp2 (D425A/C459S) and 48 h later, proteins were immunoblotted with HA, Flag, Occludin and actin antibodies (I). Reciprocal immunoprecipitations with Flag (J) or HA (K) antibodies and the interaction are examined by immunoblotting with HA (J, M), Flag (K, L) antibodies. Note: *We expressed four independent clones of HA-Shp2 (D425A/C459S) in HEK293T cells, but only two of these clones were successfully expressed.