(A) Alignment of Lem8 with several known cysteine proteases obtained by PSI-BLAST analysis. The strictly conserved catalytic residues are marked in red. Shown cysteine proteases are HopN1 and …
The strictly conserved residues were shown in dark purple background and the Cys-His-Asp motif are marked with white letters in a red background. HopN1 and AvrpphB are from P. syringae. YopT and …
(A) Subcellular distribution of Lem8. Two hours after infected with the indicated bacterial strains, bone marrow-derived macrophages (BMDMs) were immunostained using anti-Legionella antibody to …
(A) Interactions between Lem8 and 14-3-3ζ detected by yeast two-hybrid assay. Yeast strains harboring the indicated constructs were streaked on Leu− and Trp− medium to select for plasmids (left) or …
Lysates of HEK293T cells expressing indicated HA-tagged proteins were subjected to immunoprecipitation with agarose beads coated with the HA antibody. The precipitates, as well as His6-Lem8 purified …
(A) Self-processing of Lem8 requires 14-3-3ζ. His6-Lem8 or His6-Lem8C280S was incubated with His6-14-3-3ζ for 2 hr, proteins resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis …
(A) Determination of the self-cleavage site of Lem8 by mass spectrometry. A diagram of the sequence containing the recognition site with the two diagnostic peptides used to determine the cleavage …
(A) Multiple alignments of the self-cleavage site of Lem8 with potential targets in human cells identified by bioinformatic analysis. Identical residues are highlighted in red. (B) Lem8 reduces the …
(A) Cleavage of substrate candidates by Lem8. Flag- or HA-tagged Rasgrp2, Pak6, Exoc8, Ankrd13B, Chkb, Ppp6R1, Kiaa1033, Gnal, and Gpr61 expressed in HEK293T cells each was immunoprecipitated with …
(A) Lem8 harbors a putative coil motif. A predicted coiled coil motif located in the amino end of Lem8 (top panel). The sequence ranges from the 45th residue to the 73rd residue with a coiled coil …
(A) The autoprocessed form of Lem8 cleaves Phldb2 in cells. HA-Phldb2-Flag was coexpressed in HEK293T cells with Lem8 or the indicated truncation mutants including the self-processed form, Lem8∆C52. …
(A) Establishment of cell lines stably expressing Lem8 or its enzymatically inactive mutant. HEK293T cells were transduced with lentiviral particles harboring the indicated plasmid at an …
(A) Overexpression of Phldb2 in 293T cells stably expressing Lem8. A HEK293T-derived cell line stably expressing Lem8 was transfected with empty vector or HA-Phldb2 for 18 hr, and the cell lysates …
Potential Lem8 interacting proteins identified by yeast two-hybrid screenings.
Bacterial strains, plasmids and primers used in the study.
Source data for figures and figure supplements.