Prokaryotic expression of WT and mutated ALAS2 proteins. (A) Rates of ALA formation by bacterial lysates expressing either WT ALAS2, negative control with a misense mutation (C344X), a gain of function control (delAT), and Y586F-mutated ALAS2; data are expressed as mean ± SD for 3 experiments. *P < .05; **P < .001 (Student t test). (B) Coomassie blue stain of a 8% acrylamide gel with lysates of E coli cells expressing either WT or mutated ALAS2 cDNAs and run on acrylamide gel. Lysates were prepared either after the sonication step (ie, before assay) or after the assay (20 minutes at 37°C). (C) Western blot analysis of lysates of E coli cells expressing either WT or mutated ALAS2 cDNAs. After protein transfer, detection was performed by incubating the membranes with a primary antibody directed against the MBP tag. A prestained protein ladder was used as molecular weight markers (MW).