(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 antibodies specific for Flag or HA. Proteins eluted with 3× Flag or HA peptides were incubated with His-Lem8 and His6-14-3-3ζ. Samples resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were probed by immunoblotting with a Flag- or HA-specific antibody. Note that Flag- or HA-GPR61 did not express so it was not examined. Results shown were one representative from three independent experiments with similar results. (B) Lem8 causes redistribution of Phldb2 in cells. Hela cells were transfected to express the indicated mCherry fusion proteins. Twenty-four hours after transfection, cells were fixed and immunostained with anti-Phldb2 antibodies. The nuclei were stained by Hoechst 33,342. Images were acquired with a Zeiss LSM 880 confocal microscope. Phldb2, green (GFP); Lem8 and its mutants, red (mCherry); nuclei, blue (Hoechst). The percentage of Phldb2-positive cells was calculated in Lem8-positive cells (right panel). Bar, 10 μm. (C) Mutations were introduced into HA-Phldb2-Flag to replace residues Arg1111 and Gln1112 (Phldb2AA1) or Gln1112 and Arg1113 (Phldb2AA2) with alanine, respectively. The two mutants were coexpressed in HEK293T cells with Lem8 or Lem8C280S. Samples were resolved with SDS–PAGE, and probed by immunoblotting with the antibody specific to HA and Flag, respectively. Mutations in the cleavage site of Phldb2 cannot completely prevent its degradation by Lem8. Results shown were one representative from three independent experiments with similar results. (D) Lem8 removes the GFP tag fused to the amino end of Phldb2 deletion mutants. GFP was fused to the amino end of Phldb2 and the indicated truncation mutants. The fusion proteins were individually coexpressed with HA-Lem8 or HA-Lem8C280S in HEK293T cells by transfection. Samples resolved by SDS–PAGE were detected by immunoblotting with GFP-specific antibodies. Results shown were one representative from three independent experiments with similar results. (E) Cleavage of Phldb2 is undetectable during L. pneumophila infection. HEK293T cells transfected to express FcγRII receptor were infected with the indicated bacterial strains. Two hours after infection, the protein levels of Phldb2, as well as the translocation and expression of Lem8, were probed with the appropriate antibodies with Tubulin and ICDH as loading control, respectively. Results shown were one representative from three independent experiments with similar results. Bacterial strains: I, noninfection; II, Lp02 (WT); III, dotA− (defective in Dot/Icm); IV, Lp02Δlem8; V, Lp02Δlem8 (pLem8); VI, Lp02Δlem8 (pLem8C280S).