1. Fig. 1. Thorase variants show defects in Thorase oligomer and GluA2-GRIP1 complex disassembly.
Thorase variants show defects in Thorase oligomer and GluA2-GRIP1 complex disassembly. (A) Immunoblot analyses of oligomer formation by wild-type (WT) Thorase and the Thorase variants. The samples were cross-linked by glutaraldehyde in the presence of 1 mM adenosine diphosphate (ADP) or 1 mM adenosine triphosphate (ATP). The ATP-treated samples were incubated at 4°C for Thorase oligomer formation and then at 37°C for ATP hydrolysis to enable disassembly of the Thorase oligomers. Samples were collected every 15 min for 150 min (lanes 1 to 10) during the incubation for cross-linking. (B) Graphical representation of the percentages of the oligomer states of Thorase in different fractions collected at 15-min intervals. (C) Immunoblot analyses of FLAG-tagged Thorase immunoprecipitations (IP) of mouse primary cortical neurons from heterozygous Thorase-deficient mice expressing WT Thorase or the Thorase variants in the presence of different nucleotides. The samples were resolved on 10% SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with anti-Thorase antibody, anti-FLAG–horseradish peroxidase antibody, anti-GluA2 antibody, or anti–glutamate receptor-interacting protein 1 (GRIP1) antibody. (D) Normalized percent bound GluA2 and GRIP1 in the Thorase FLAG-tagged immunoprecipitated samples in (C). (E) Immunoblot analyses of GluA2 immunoprecipitation of mouse primary cortical neurons from heterozygous Thorase-deficient mice expressing FLAG-tagged WT Thorase or the Thorase variants in the presence of different nucleotides. The samples were separated on 10% SDS-PAGE and immunoblotted with anti-FLAG antibody, anti-GluA2 antibody, or anti-GRIP1 antibody. (F) Normalized percent bound Thorase and GRIP1 in the GluA2 immunoprecipitated samples in (E). (G) Percent of Thorase and GRIP1 disassembled (upon ATP hydrolysis) from the GluA2 complexes in (E). Means ± SEM (n = 3). *P < 0.10; n.s. (nonsignificant), P > 0.10, Holm-Sidak post hoc test compared with WT. Power (1 − β error probability) = 0.8 to 1.0.
George K. E. Umanah et al., Sci Transl Med 2017;9:eaah4985
Published by AAAS