Fig. 3: UFL1 ufmylates AKT1 at Lys189, Lys276, and Lys297.
From: Targeting the UFL1-AKT cascade suppresses triple-negative breast cancer progression
a In vivo UFMylation assay of AKT1 in HEK293T cells transfected with Flag-S-AKT1 and the components of the UFMylation system. b In vitro UFMylation assay of AKT1. Samples were subjected to western blotting with an anti-UFM1 antibody. c In MDA-MB-231 and HCC1806 cells stably expressing control or UFL1 shRNA, UFMylation of endogenous AKT1 was detected by immunoprecipitation with an anti-AKT1 antibody, followed by subsequent western blotting using an anti-UFM1 antibody. d A schematic showing the generation of the UFL1 full-length (FL), 1–247 aa and 248-C aa constructs. e Cells were transfected with vector, HA-S-UFL1 full length or HA-S-UFL1 truncation mutants (1–247 aa and 248-C aa). Cell lysates were pulled down using S-agarose, and the interaction between UFL1 and AKT1 was then examined. f A schematic showing the generation of the AKT1 full-length (FL), 1–148 aa, 148–410 aa and 410–C aa constructs. g Cells were transfected with vector, Flag-S-AKT1 truncation mutants (1–148, 148–410 and 410-C). Cell lysates were pulled down using S-agarose, and the interaction between UFL1 and AKT1 was then examined. h UFMylation assay of the Flag-S-AKT1 full length and its truncation mutants (1–148 aa, 148–410 aa or 410-C aa) was examined in HEK293T cells. i, j UFMylation assay of the HA-S-AKT1 WT and 3KR was performed in MDA-MB-231 and HCC1806 cells. Source data are provided as a Source Data file.
