Figure 6.

Western blot analyses of patient and control skin fibroblast lysate and supernatant under reducing conditions. Patients 1–3 carry C1r p.V50D (CUB1), patient 4 carries C1r p.R301P (CUB2), and patient 5 carries C1r p.C309F (CCP1). Detection with C-terminal C1r antibody in cell extracts (A) and supernatant (B), and with N-terminal C1r (C) and C1s (D) antibodies in supernatant. Control cells showed uncleaved full-length C1r and C1s protein both within cells (A) as well as supernatant (B–D), with little evidence of activation. In patient samples, both full-length and activated C1r (B-chain) were detected in patient cell extracts, but no C1r protein bands were visible in supernatants except at low amounts in patient 5. In contrast to controls, no full-length C1s was present in supernatants of all patient fibroblasts (D) indicating complete C1s activation caused by the presence of heterozygous C1r variants. Additional bands in patients 2 and 5 cell extracts (~80 kDa), and control 2 and patient 5 supernatants (~115 kDa) with the C-terminal C1r antibody are non-specific.