In silico and in vitro characterization of hACTN4-M240T stability. (a) In silico prediction of M240T mutation on alpha-actinin protein stability (delta-delta-G) using 4 different computational algorithms. M>T amino acid exchange in the conserved domain has a destabilizing effect in ACTN4 as well as ACTN1 and ACTN3. (b) Representative western blot analysis of whole-cell lysates cotransfected with Flag-hACTN4-WT or Flag-hACTN4-M240T and Flag-GFP serving as expression control. (c) Densitometric quantification of 3 independent experiments as shown in (b). Compared with the WT variant, hACTN4-M240T is significantly lower expressed (n = 3, error bars indicate SD, ∗P < 0.05, two-tailed t test). GFP, green fluorescent protein; WT, wild-type.