Figure 4. Reelin is cleaved within its CTR by PC family proteases. A, Schematic of Reelin protein and amino acid sequence of the CTR. Boxed region highlights the consensus sequence for PC family proteases. Arrows indicate two known cleavage sites. Arrowheads indicate epitopes of anti-Reelin antibodies. B, Schematic of Reelin mutants with a FLAG-tag. Solid black boxes represent FLAG-tags. C, Secreted ReelinC-FLAG did not retain its FLAG-tag. The culture supernatants of HEK293T cells expressing the above Reelin mutants were analyzed by WB with the indicated antibodies. The reactivity of ReelinFLAG+C to anti-FLAG antibody is weaker than ReelinΔC-FLAG for unknown reason (Kohno et al., 2009b). D, Intracellular ReelinC-FLAG retained the FLAG-tag. The culture supernatants and whole-cell lysates of ReelinC-FLAG-expressing HEK293T cells were analyzed by WB. E, Reelin is cleaved by a PC family protease(s). The culture supernatants from ReelinC-FLAG-expressing HEK293T cells (lanes 1–3) or COS-7 cells (lanes 4–6) cultured without inhibitors (lanes 1 and 4), with FI-1 (lanes 2 and 5), or FI-2 (lanes 3 and 6) were analyzed by WB with G10 (top) or anti-FLAG (bottom). Asterisks indicate full-length Reelin. Arrowheads indicate Reelin fragments generated by cleavage within RR3 and cleavage between RR6 and RR7, respectively. FI-1 inhibits cleavage at both sites (Kohno et al., 2009a). Secreted ReelinC-FLAG protein retained the FLAG-tag in the presence of the PC inhibitors (lanes 2, 3, 5, and 6). F, LoVo cells, which bear no endogenous PC activity, do not cleave Reelin at the WC site. The expression vector for ReelinC-FLAG or ReelinC-Venus was transfected into LoVo cells with or without a furin vector. The culture supernatant was collected and analyzed by WB with the indicated antibodies. G, Schematic of AP-RR78CF and its mutants. The consensus sequence of PC family proteases is underlined. Mutated amino acids are shown in bold. H, R3455 is necessary for cleavage by PC family proteases. The culture supernatants of HEK293T cells expressing the indicated proteins with or without FI-1 were analyzed by WB. I, Monoclonal antibodies 12C10 and 1D4 specifically recognize ReelinFL. Wild-type Reelin (WT), ReelinC-FLAG, or a mutant Reelin lacking the last 6 residues (Δ6) were expressed in HEK293T cells cultured with or without FI-1. The culture supernatants were analyzed by WB with the indicated antibodies. J, FI-1 inhibits WC cleavage by CGNs. CGNs were cultured with the indicated inhibitors, and the culture supernatants were analyzed by WB. ReelinWT and ReelinΔ6 from HEK293T cells were run as references. K, The ratio of endogenous Reelin protein cleaved at the WC site is dependent on developmental stage. Reelin proteins were immunoprecipitated using anti-Reelin CR-50 antibody from the brain homogenate at the indicated stages, analyzed by WB, and quantified. Error bars indicate mean ± SEM. F(2,6) = 19.42. **p < 0.01 (Tukey's test). N.S., Not significant. n = 3. L, WC cleavage had little effect on the ability of Reelin to induce Dab1 phosphorylation in cultured cerebral cortical neurons in the standard assay. Cerebral cortical neurons were incubated for 20 min at 37°C with conditioned medium containing ReelinWT collected from HEK293T cells in the presence of FI-1 (ReelinFL) or ReelinΔ6 collected in the presence of FI-1. Whole-cell lysates were separated by SDS-PAGE followed by WB using anti-phosphotyrosine 4G10 (top) and anti-Dab1 (bottom) antibodies. Molecular mass markers (kDa) are shown on the left of the panel. Error bars indicate mean ± SEM. F(2,6) = 50.21. **p < 0.01 (Tukey's test). N.S., Not significant. n = 3. M, ReelinFL localizes to the MZ. Sections of the cerebral cortices of wild-type (+/+, top) or reeler (rl/rl, bottom) mice at P1 were immunostained with AF3820 (antibody against the N-terminal region of Reelin, red) and 12C10 (green). Nuclei were stained with Hoechst 33342 (blue). In sections from wild-type mice, the 12C10 antibody stained Cajal–Retzius cells in the MZ. Arrowheads indicate signals positive for AF3820 only. Scale bar, 50 μm. F, I, K, Only the bands of full-length Reelin protein (430 kDa) are shown.