Figure 5.

CAP2 forms dimers through Cys³². (A) Co-immunoprecipitation assays carried out from homogenate of HEK293 cells transfected with EGFP-CAP2 and either Myc-CAP2 or Myc-CAP2(165-476). The samples derive from the same experiment and gels/blots were processed in parallel. Full uncropped blots are available in the Supplementary material. (B) The analysis of the optical density (OD) reveals that the lack of the sequence 1-164 of CAP2 significantly reduces CAP2 self-association [Myc-CAP2(165-476)/EGFP-CAP2 versus Myc-CAP2/EGFP-CAP2, paired t-test P = 0.036, n = 4 independent experiments]. (C) Representative WB of homogenates of rat brain hippocampus loaded onto a non-denaturating gel, without (left side) or with dithiothreitol (DTT) treatment (right side), as indicated. The band corresponding to the dimeric form of CAP2 is eliminated by DTT treatment. Full uncropped blots are available in the supplementary material. (D) Representative WB of homogenates of HEK293 cells expressing Myc-CAP2, Myc-CAP2(165-476) or Myc-(C32G)CAP2. The deletion of the 1-164 region or the single point mutation Cys³² to Gly avoids the capability to form dimers. The samples derive from the same experiment and gels/blots were processed in parallel. Full uncropped blots are available in the supplementary material. (E) Representative confocal images of PLA experiments showing proximity between EGFP-CAP2 and either Myc-CAP2 or Myc-(C32G)CAP2 (white) along MAP2 positive dendrites (magenta); lower panels, inverted images of PLA signal (black), scale bar = 5 μm. (F) Graphs show the quantification of the PLA clusters/μm (EGFP-CAP2/Myc-CAP2 = 1.366 ± 0.150, EGFP-CAP2/Myc-(C32G)CAP2 = 0.566 ± 0.117, t-test from mixed-effects model: ***P < 0.0001; n EGFP-CAP2/Myc-CAP2 = 16 neurons, n EGFP-CAP2/Myc-(C32G)CAP2 = 16 neurons).