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. 2018 Jan 19;13(1):e0191224. doi: 10.1371/journal.pone.0191224

Fig 4. Reduced secretion and intracellular and extracellular aggregation of Arg446Cys VWA2.

Fig 4

(a) Cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the One-STrEP-tag. Arrowheads indicate monomeric VWA2. On the right, equal loading is demonstrated by Ponceau staining of the membranes. Asterisks indicate artefact bands. (b) cDNA from non transfected (nt), non-transfected ER stress induced (nt+DTT), wt VWA2 transfected (wt) and Arg446Cys VWA2 (R446C) transfected 293EBNA cells was submitted to RT-PCR and the PCR products separated by agarose gel electrophoresis. Arrows indicate the bands for XBP-1 and ER stress induced XBP-1s. Equal loading is demonstrated by actin control RT-PCR shown below. (c) Equal amounts (0.2 μg) of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the C-terminal fragment (P3) of human VWA2. Under non-reducing conditions higher aggregates are seen. Arrows indicate the border between separation and stacking gel. (d) Equal amounts of cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells as in (a) and of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) as in (b) were separated by agarose-polyacrylamide composite gels under non-reducing conditions and detected with an antibody against the One-STrEP-tag.