Fig. 2: Expression and localization of CTRP5 in L-ORD-iRPE.

a, b Apical and basal CTRP5 secretion measured by ELISA in the culture medium are both significantly decreased (n = 14). c Coexpression of V5-tagged WT CTRP5 (green) and FLAG-tagged S163R CTRP5 (red) in healthy-iRPE. V5-tagged WT CTRP5 expressing lentivirus construct was transduced at MOI 0.5 for both top and bottom panels. MOI of Flag-tagged S163R CTRP5 expressing lentivirus construct was 0.5 for cells in the top panel and 3.0 for cells in the bottom panel. Scale bar: 10 µm d Confocal microscopy images of untreated and bafilomycin (BafA1) treated (3 h) healthy-iRPE and L-ORD-iRPE co-stained with CTRP5 (red) and ATG5 (green). (n = 3 images per condition). Scale bar: 10 µm. e Representative confocal microscopy images showing colocalization of CTRP5 (red) with membrane receptors ADIPOR1 (green, upper panel) and no colocalization with ADIPOR2 (green, lower panel). Nuclear stain (blue). Scale bar: 5 µm. f TEM image of immunogold labeled ADIPOR1 (6 nm gold particle) and CTRP5 (12 nm gold particle) demonstrate the co-binding of two proteins (arrow). Scale bar: 500 nm. g Western blot detects CTRP5 in the membrane fraction of iRPE cells immunoprecipitated using anti-ADIPOR1 antibodies and not in lanes immunoprecipitated with the IgG antibody or with only beads. h Probabilistic model of the interaction of integral membrane protein ADIPOR1 (blue) and CTRP5 (teal) determined using published crystallographic structures and refined by molecular dynamics. i The polar serine to arginine substitution on CTRP5 (blue) is predicted to have an electrostatic repulsive interaction with neighboring arginine residue, Arg122, on ADIPOR1 (magenta) reducing the likelihood of interaction. See also Fig. S4. ∗∗∗p < 0.001.