Fig. 5: Calcineurin interacts with Parkin. | Cell Death & Differentiation

Fig. 5: Calcineurin interacts with Parkin.

From: Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy in flies

Fig. 5

A In vitro interaction assay: schematic representation. Affinity-purified recombinant His-tagged Parkin is produced from bacteria, and coupled to a His-affinity resin to generate Parkin-His-resin. The resin is incubated with protein lysate extracted from cells expressing Flag-tagged CaN. The resin is washed to remove nonspecifically adhering proteins, and Imidazole is used to elute the complexes from the resin. The obtained eluate is separated by SDS-PAGE and analyzed by immunoblotting. B In vitro interaction assay. His-tagged Parkin or His-tagged MEF2D coupled to a His-affinity resin are incubated with cell lysate obtained from MEFs transfected with CaN-Flag or USP14-Flag, as indicated. The eluate is separated by SDS-PAGE and analyzed by immunoblotting using anti-Flag, anti-Parkin and anti-His antibodies (FT = flow through; EL=eluate). C Representative images of Proximity Ligation Assay (PLA) performed on HeLa cells transfected with Parkin-mCherry and CaN-FLAG (PPP3CB-FLAG). After fixation, cells were processed for investigating Parkin proximity interactions by incubating with anti-Parkin antibody and anti-PPP3CB antibody, and corresponding secondary antibodies, or secondary antibodies alone as negative control. CaN and Parkin interactions are represented by green dots. As negative biological control for PLA we used anti-Parkin antibody and anti-GRASP65 antibody in YPF-Parkin expressing HeLa cells. As positive control for PLA, we used anti-GRASP65 antibody and anti-GM130 antibody. GRASP65 is a peripheral membrane protein that resides in the cis-Golgi apparatus, and interacts with GM130 (also expressed in the cis-Golgi network) but not with Parkin. GRASP65 and GM130 interactions are represented by red dots. White squares contain higher-magnification images. D Quantification of C. Graph bar shows mean ± SEM of PLA dots per cell for at least ≥ 350 cells per biological replicate. Student’s t-test (n = 3-4; p < 0.05). E HEK 293 T cells were treated with DMSO or CCCP-2 h and subjected to immunoprecipitation (IP) of Parkin using mouse anti-Parkin antibody or anti-mouse IgG as negative control. Western Blot analysis was performed with rabbit anti-CaN antibody or mouse anti-Parkin antibody on the pulled-down samples. Inputs represent 5% of the protein lysates and IP eluate 100% of the protein lysates. Mouse IgG TrueBlot® ULTRA enabled detection of Parkin band, without hindrance by interfering immunoprecipitating IgG heavy chains.

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