Expression and thermostability of the Ca²⁺-binding site mutants in A2∆CC. (A) VWF A2 domain fragments with (A2VicCC) and without (A2∆CC) the vicinal cysteines were transiently expressed in HEK293EBNA cells. After 3 days, the medium was collected and the cells lysed with PBS 1% igepal and vigorous pipetting. After centrifugation to remove cell debris, the medium and lysate were run on 4% to 12% Bis-Tris gels and the VWF A2 domain protein detected on western blot with an antibody against the C-terminal myc tag. (B-C) The successfully secreted A2∆CC WT and Ca²⁺ mutants were subsequently purified and subjected to DSF analysis. Results are means ± SEM of at least 3 independent experiments.