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. 2021 Apr 15;12:607641. doi: 10.3389/fimmu.2021.607641

Figure 4.

Figure 4

Truncated Sez6L2 inhibits alternative pathway hemolysis more than classical pathway hemolysis. (A) Schematic of Sez6L2 and Sez6L2-MH domain structures. CCP=Domain abundant in complement control proteins. CCP domains are also known as SUSHI repeats or short complement-like repeat (SCR). CUB= Domains named after complement C1r/C1s, uEGF, and BMP1. TM=Transmembrane region. Sez6L2-MH was made by replacing the transmembrane and cytoplasmic tail domains with a tandem Myc, 6xHis tag. (B) Purified Sez6L2-MH is shown by a Coomassie stained gel and by western blot with anti-Sez6L2 and anti-Myc antibodies. Lanes with the Coomassie stain are from the same gel. (C) Classical pathway hemolysis assay. Antibody-coated sheep erythrocytes were exposed to human serum pre-incubated with purified Sez6L2-MH, H-DAF, FH, C4BP, C1-INH, or BSA. After 30 mins, the percent of cell lysis was measured by spectrophotometry (A415). One-way ANOVA (P <0.0001; F(6,30)=233.2); PBS N=10; Sez6L2-MH N=7; H-DAF N=4; CFH N=4; C4BP N=3; C1-INH N=3; BSA N=6. (D) Alternative pathway hemolysis assay. Rabbit erythrocytes were exposed to human serum pre-incubated with Sez6L2-MH, complement regulators, or BSA in presence of 10 mM MgEGTA to block the classical pathway. Then the percent of cell lysis was measured by spectrophotometry (A415). One-way ANOVA (P <0.0001; F(4,16)=33.88). PBS N=6, Sez6L2-MH N=5; FH N=2, H-DAF N=2, and BSA N=6. For all graphs *p < 0.05 compared to PBS control and #p < 0.05 compared to BSA negative control.