Fig. 3 TBK1 phosphorylates RAB7AS72 but not the equivalent residue in other RABs in vitro. TBK1 phosphorylates RAB7AS72 but not the equivalent residue.

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Fig. 3 TBK1 phosphorylates RAB7AS72 but not the equivalent residue in other RABs in vitro. TBK1 phosphorylates RAB7AS72 but not the equivalent residue in other RABs in vitro. (A) Coomassie blue PAGE analysis of purified GST-RAB7AWT and RAB7AS72A after purification from E. coli. (B) The indicated GST-RAB7A proteins were incubated with recombinant TcPINK1 (E. coli) (27) or GST-TBK1 (insect cells) (1 hour at 30°C, with or without ATP), and reaction products were separated by Phostag-PAGE before immunoblotting with α-RAB7A antibody. (C) Alignment of the amino acid sequence around S72 in RAB7A (red asterisk) and selected RABs. (D) Coomassie blue SDS-PAGE analysis of purified GST-RAB1A, GST-RAB1B, and GST-RAB7L (with or without mutation of the S72 equivalent to Ala) after purification from E. coli. (E) The indicated GST-RABs were incubated with or without GST-TBK1 (1 hour at 30°C, with ATP), and reaction products were separated by Phostag-PAGE and immunoblotted with α-GST antibody. (F) Coomassie blue SDS-PAGE analysis of purified GST-RAB8AWT, RAB8AS72A, and RAB8AS111A after purification from E. coli. (G) The indicated GST-RAB8A proteins were incubated with or without GST-TBK1 and analyzed as in (E). J.-M. Heo et al. Sci Adv 2018;4:eaav0443 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).