Fig. 5: Fibrinogen chains accumulate and form insoluble aggregates in Sel1L-deficient livers.

a The overall structure of fibrinogen showing a hexamer of Aα, Bβ and γ chains. The 29 highly conserved disulfide bonds are shown as red bars, and the glycosylation sites on Bβ and γ are marked in purple. Aα is not glycosylated. Image created in BioRender. Tushi, N. (2024) BioRender.com/l49l811. (b–e) Liver samples from 6-week-old WT and Sel1L^Alb littermates were analyzed for: (b) reducing Western blot analysis with quantitation normalized to HSP90 shown in (c); (d) qPCR analysis; (e) non-reducing SDS-PAGE and Western blot analysis using the same protein lysates shown in (b). In (b) and (e), fibrinogen proteins were analyzed by antibodies specific for each chain or all fibrinogen chains (Fib). Red arrows point to high molecular weight aggregates of fibrinogen. N = 3 for Western blot analyses, n = 5 for WT and n = 6 for Sel1L^Alb for qPCR analyses. Values, mean ± SEM. *, p < 0.05; **, p < 0.01 by two-tailed Student’s t test. f Sucrose gradient fractionation (fractions 1 to 11 from top to bottom) of liver samples from WT and Sel1L^Alb littermates analyzed by nonreducing or reducing SDS-PAGE using an Aα chain-specific antibody. Red arrow points to fibrinogen aggregates. #, a non-specific band. Quantitation of reduced Aα is shown on the right with fractions with Aα monomers, assembly intermediates and high molecular weight (HMW) aggregates labeled. HSP90, a loading control. g Liver samples from 6-week-old WT and Sel1L^Alb littermates were prepared in lysis buffer containing 0.5% Nonidet P-40 (NP40) and analyzed for protein distribution in the NP40 soluble and NP40 insoluble (pellet) fractions by Western blot. The distribution of HSP90 and H2A marks the soluble and insoluble fractions, respectively. Quantitation of fibrinogen chains normalized to H2A in NP40 insoluble fraction is shown below the blots. (f–g), representative data from two independent repeats. Source data are provided as a Source Data file.