![Figure 6. LRCH1 attenuates DOCK8-mediated Cdc42 activation for T cell migration. (A) T8.1 cells were transfected with FLAG-DOCK8, incubated with Ttox peptide-pulsed L625.7 cells to form cell conjugates, followed by immunostaining to visualize DOCK8 and Cdc42. Bar, 5 µm. (B) The FRET efficiency of biosensor Raichu-Cdc42 between WT and Lrch1−/−CD4+ T cells in response to SDF-1αtreatment. FRET efficiency was measured with donor dequenching approach, and was calculated as E = (Post −[Pre/Post]) × 100%, where Post and Pre represents the donor fluorescence before and after photo bleaching (left). n = 20. NS, not significant (P > 0.05); **, P < 0.01. Activated Cdc42 was precipitated by a GST-CRIB-PAK1 pull-down assay in T8.1 cells that overexpressed LRCH1 or the vector control (right). (C) 293T cells were transfected with FLAG-DOCK8 with or without HA-LRCH1, and the amount of DOCK8 binding to the GST-Cdc42G15A–coated beads was used to evaluate the GEF activity of DOCK8. (D) 293T cells were cotransfected with FLAG-DHR-2, HA-Cdc42 with or without HA-LRCH1. The cell lysates were subjected to a GST-CRIB-PAK1 pull-down assay to precipitate the active Cdc42. (E) 293T cells were transfected with HA-Cdc42, Myc-LRCH1, and FLAG-DHR-2, followed by immunoprecipitation with anti-HA and immunoblotting with anti-HA or anti-FLAG. (F) The purity of the purified His-DHR-2 (1632–2068 aa) and GST-Cdc42 G15A protein from E. coli was determined by Coomassie blue staining. (G and H) Increasing amounts of FLAG-LRR1-9 (G) or FLAG-L305-763 (H) were added into the solution containing the purified recombinant proteins His-DHR-2 and GST-Cdc42G15A, incubated and then subjected for precipitation using anti-His antibody. (I) T8.1 cells, which were transfected with FLAG-DOCK8 and HA-LRCH1, were treated with or without SDF-1α to assess localization of DOCK8 (red, top) and LRCH1 (green, middle). Bar, 5 µm. (J and K) T8.1 cells expressing FLAG-DOCK8 and HA-LRCH1 were treated or untreated with SDF-1α and PMA (J). 293T cells were transfected with HA-LRCH1, FLAG-DOCK8, or the membrane-localized CVIM-FLAG-DOCK8 (K), followed by immunoprecipitation with anti-FLAG to detect the phosphorylation levels of DOCK8 and the amount of LRCH1. Data presented are representative of two independent experiments (A–D, G, H, and J), or three independent experiments (E, I, and K). Intensity of the immunoblots was quantified and shown at the bottom (mean ± SD). Statistical significance was determined using unpaired Student’s t test.](https://cdn.rupress.org/rup/content_public/journal/jem/214/1/10.1084_jem.20160068/8/jem_20160068_fig6.jpeg?Expires=1767669827&Signature=LlxATryr0Vdp6mS1hERt~3NhsPJSmeSkd-lXYP8XuCRlpaYkGjdKO4aW4LOM4N6LQuIF-z5Dw93Ct4aqwiQTske348bXBs9L2oXrx-xRZDkFlozCDz1U~UWUWrarZLzqNL2UbpNlbvvsp52npAKUGXy-hrORAMeJrwuia4pjQow8Xk4RK37AGsszpOdXnLLI7K9E~WGpltK6s8LeouXy7XAcRlBuB-0XX7XAB4IU5xFerJg5s2KoRIS6rkdCongnR7RBH0hcaHbXs3Hlz9-BocLGAg5408hgxZ-xpENWcIkZDF3EF2E~rRYiY-QX2QFC6diAu2DZmO522GgISwrkDg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
LRCH1 attenuates DOCK8-mediated Cdc42 activation for T cell migration. (A) T8.1 cells were transfected with FLAG-DOCK8, incubated with Ttox peptide-pulsed L625.7 cells to form cell conjugates, followed by immunostaining to visualize DOCK8 and Cdc42. Bar, 5 µm. (B) The FRET efficiency of biosensor Raichu-Cdc42 between WT and Lrch1−/−CD4+ T cells in response to SDF-1αtreatment. FRET efficiency was measured with donor dequenching approach, and was calculated as E = (Post −[Pre/Post]) × 100%, where Post and Pre represents the donor fluorescence before and after photo bleaching (left). n = 20. NS, not significant (P > 0.05); **, P < 0.01. Activated Cdc42 was precipitated by a GST-CRIB-PAK1 pull-down assay in T8.1 cells that overexpressed LRCH1 or the vector control (right). (C) 293T cells were transfected with FLAG-DOCK8 with or without HA-LRCH1, and the amount of DOCK8 binding to the GST-Cdc42G15A–coated beads was used to evaluate the GEF activity of DOCK8. (D) 293T cells were cotransfected with FLAG-DHR-2, HA-Cdc42 with or without HA-LRCH1. The cell lysates were subjected to a GST-CRIB-PAK1 pull-down assay to precipitate the active Cdc42. (E) 293T cells were transfected with HA-Cdc42, Myc-LRCH1, and FLAG-DHR-2, followed by immunoprecipitation with anti-HA and immunoblotting with anti-HA or anti-FLAG. (F) The purity of the purified His-DHR-2 (1632–2068 aa) and GST-Cdc42 G15A protein from E. coli was determined by Coomassie blue staining. (G and H) Increasing amounts of FLAG-LRR1-9 (G) or FLAG-L305-763 (H) were added into the solution containing the purified recombinant proteins His-DHR-2 and GST-Cdc42G15A, incubated and then subjected for precipitation using anti-His antibody. (I) T8.1 cells, which were transfected with FLAG-DOCK8 and HA-LRCH1, were treated with or without SDF-1α to assess localization of DOCK8 (red, top) and LRCH1 (green, middle). Bar, 5 µm. (J and K) T8.1 cells expressing FLAG-DOCK8 and HA-LRCH1 were treated or untreated with SDF-1α and PMA (J). 293T cells were transfected with HA-LRCH1, FLAG-DOCK8, or the membrane-localized CVIM-FLAG-DOCK8 (K), followed by immunoprecipitation with anti-FLAG to detect the phosphorylation levels of DOCK8 and the amount of LRCH1. Data presented are representative of two independent experiments (A–D, G, H, and J), or three independent experiments (E, I, and K). Intensity of the immunoblots was quantified and shown at the bottom (mean ± SD). Statistical significance was determined using unpaired Student’s t test.
