Figure 1
C-terminal truncation decreases CAPS-1 synaptic enrichment. (a) Schematic representation of mouse CAPS-1, showing the C2 ___domain (397–516)16, 17, PH ___domain (516–632)16, 17, MHD ___domain (933–1113)19, 20, and DCV ___domain (1220–1355)16. Numbers represent amino acid residues. Indicated are the CAPS-1 C2 ___domain mutants (K428E and G476E), the CAPS-1 PH ___domain mutant (R558D/K560E/K561E, RKK) and C-terminal truncation (Δ654–1355, ΔC). (b) Western blot of CAPS DKO cortical neurons infected with wild type or mutant CAPS-1 constructs and CAPS-2 KO control neurons (control). Actin was used as loading control, gel was cropped (full-length gel presented in Figure S2). (c) Quantification of relative CAPS-1 level in control neurons of two independent western blots of CAPS DKO cortical neurons infected with wild type or mutant CAPS-1 constructs and control neurons. CAPS-1 level was corrected for protein loading (using actin levels). (d) Representative images of CAPS DKO hippocampal neurons infected with WT, K428E, G476E, RKK, ΔC and control neurons, stained with dendrite marker (MAP2, blue), CAPS-1 (magenta) and synaptophysin 1 (syph, green). Boxed areas are enlarged at the bottom. (e) Manders’ coefficient of CAPS-1 in synaptophysin (syph), relative to colocalization of VAMP in syph, in CAPS DKO hippocampal neurons infected with wild type or mutant CAPS-1 constructs and control neurons. One-way ANOVA (CAPS conditions): p = 0.067 (not significant, ns). (f) Number of CAPS-1 puncta in control and DKO + ΔC neurons. Detailed information (average, SEM, n and detailed statistics) is shown in Table S1.
