Figure 1.

K8 Q70N mutation inhibits TG2-mediated cross-linking. A) Schematic of the human K8 protein domains showing the location of glutamine (Q) and lysine (K) residues in the non-α-helical K8 head and tail domains. B) Extracts from untransfected BHK cells and cells transfected with WT K18 plus WT K8 or the K8 mutants: Q7N, Q70N, Q85N, Q85N/Q90N, and Q408N were separated on SDS-PAGE then blotted with antikeratin antibodies. C) Extracts from BHK cells transfected with WT K18 plus WT K8 were treated with the indicated concentrations of TG2 at 37°C for 5, 15, 30, and 60 min. Reactions that lacked either TG2 or calcium were incubated at 37°C for 30 min, then quenched using 4× Laemmli sample buffer and blotted with anti-K8 antibodies. Arrows highlight the K8-containing cross-links that form in TG2- and calcium-dependent manners. D) Extracts of the cell transfectants were treated with TG2 for 15 min (37°C) or left untreated. Reaction mixtures were separated using SDS-PAGE then blotted with antibodies to K8. Arrows indicate K8-containing cross-links whose formation decreased dramatically on K8 Q70N mutation.