Figure 3: GIT1 is involved in GRK6-mediated apoptotic engulfment.
From: GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance
(a) NIH3T3 cells (4 × 104 cells) infected with the indicated retroviruses were co-cultured at 37 °C for 90 min with 1 × 106 cells CMFDA-labelled WT thymocytes. Percentages of the phagocytes carrying the engulfed thymocytes were determined by FACS. The experiments were done at least three times, and the average numbers are shown with s.e.m. (b) Schematic representation of GIT1 and its deletion mutants, GIT1 (ΔC) and GIT1 (ΔAC), used in this study. The numbers indicate the positions of the amino acids. (c) The activation state of Rac1 in NIH3T3 cells infected with the indicated retroviruses was measured in GTP-Rac1 pull-down assays with GST-CRIB. Data represent means±s.e.m. (error bars) of four independent experiments. (d) HEK293 cell lysates transiently transfected with the indicated plasmids were immunoprecipitated with anti-Flag antibody. Western blot was performed with anti-GIT1 antibody. (e) Cell extracts of BMDM from WT mice were immunoprecipitated with normal rabbit IgG or anti-GRK6 antibody. The total lysates and immunoprecipitates (IPs) of BMDMs were immunoblotted (IB) with anti-GRK6 and anti-GIT1 antibodies. (f) NIH3T3 cell lysates infected with the indicated retroviruses were immunoprecipitated with anti-Flag antibody. The IPs were subjected to western blot analysis with anti-GRK6 and anti-GIT1 antibodies. All the immunoprecipitation experiments were done at least three times, and representative data are shown. ***P<0.001, ###P<0.001.
