Figure 7.

Bortezomib partially re-addresses podocin^R138Q to the plasma membrane. A, immunofluorescence analysis showing Pod^R138Q (green) in the presence of filopodia, through colocalization with the PM marker WGA (red), after a 2-h treatment with Bz (0.1 μm) or vehicle (DMSO). Pod^wt co-localization with WGA is included as a positive control. Hoechst nuclei labeling (H) is also shown (blue). Scale bar = 10 μm. Image analysis of the WGA co-localization with podocin only at filopodia was obtained through the quantification of regions of interest (ROIs) that carefully delimit cell perimeters. Graph corresponds to the quantification of one representative experiment. One-way analysis of variance followed by Dunnett's post-test was used as statistical analysis. **, p < 0.01 and ***, p < 0.001. B, co-immunoprecipitation (IP HA) of HA-podocin and Cnx after Bz addition (16 h at 0.1 μm). The graph represents the densitometry quantification of Cnx when podocin is immunoprecipitated. Data are normalized to total immunoprecipitated podocin (HA triplet) and represent to at least three independent experiments. ***, p < 0.001. C, schematic summarizing the influence of different treatments on Pod^R138Q topology and subcellular localization. Red and green arrows indicate a dynamic change to the transmembrane “wrong” or hairpin “right” topology, respectively. Numbers circled in yellow: (1) there is an imbalance of hairpin Pod^R138Q toward a transmembrane topology, (2) decreasing Pod^R138Q interaction with Cnx through siCnx transfection favors the hairpin topology, (3) stabilization of Pod^R138Q interaction with Cnx, through inhibition of Cnx cycle exit with Kif, enhances podocin transmembrane topology, and (4) inhibition of Pod^R138Q proteasomal degradation with Bz increases the proportion of hairpin Pod^R138Q and is the only treatment that allows partial relocalization to the PM. EC, extracellular matrix; PM, plasma membrane; C, cytosol.