Fig. 2

JOSD2 depletion activates the LKB1/AMPK pathway and LKB1 kinase activity. a IPs by Streptavidin magnetic beads from 293T cells transfected with Vector or JOSD2-SBP for 48 h were subjected to AP-MS analysis of JOSD2-interacting proteins. b NCI-H1299 cells were infected with lentiviruses of scramble control or JOSD2 shRNA for 96 h and the cell lysates were subjected to RNA sequencing analysis of related pathways regulated upon JOSD2 depletion. c NCI-H1299 cells were infected with lentiviruses of scramble control or JOSD2 shRNA (#1, #2) for 96 h and the cell lysates were subjected to IB. d Immunoprecipitates (IPs) by HA beads from 293FT cells transfected with the vector or JOSD2-HA along with LKB1-Flag or AMPKa1-Flag for 48 h were subjected to IB. e IPs by anti-LKB1 antibody from NCI-H1299 cells expressing scramble control or JOSD2 shRNA (#1, #2) were subjected to in vitro kinase assay followed by IB. f The position of cysteine 24, histidine 125 and aspartic acid 140 in human JOSD2 (PDB: 6PGV). g Schematic illustration of human JOSD2 protein marked with two candidate catalytic sites including cysteine 24 (C24) and histidine 125 (H125) (top). Sequence alignment of C24 and H125 catalytic sites within JOSD2 orthologues in different species (bottom). h In vitro ubiquitin-AMC assay by incubating purified GST, GST-JOSD2 or the indicated catalytic-inactive GST-JOSD2 protein with ubiquitin-AMC as the substrate at 37 °C for 1 h. i IPs by Flag beads from 293FT cells transfected with LKB1-Flag for 48 h were incubated with purified GST, GST-JOSD2 or the indicated catalytic-inactive GST-JOSD2 proteins at 37 °C for 1 h, and then subjected to in vitro kinase assay followed by IB