Figure 2: GRK6 regulates engulfment of apoptotic cells via Rac1.
From: GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance
(a,b) NIH3T3 cells were infected with retroviruses encoding WT GRK6 and DN Rac1 (T17N) (a), or kinase-inactive GRK6 (K215R) and WT Rac1 (b), either alone or in combination. The infected cells were subjected to the engulfment assay with CMFDA-labelled WT thymocytes. Percentages of the phagocytes carrying the engulfed thymocytes were determined by FACS. (c) NIH3T3 cells infected with the empty retrovirus vector or the virus carrying the cDNA for WT GRK6 were subjected to GTP-Rac1 pull-down assay using GST-CRIB. The pull-down samples were subjected to western blot using anti-Rac1 antibody and the level of GTP-bound Rac1 was quantified by densitometry, normalized against the total amount of Rac1 in the cell lysates. Data represent means±s.e.m. (error bars), n=3. (left panel) (d,e) NIH3T3 cells were infected with retroviral combinations of WT GRK6 and two DN forms of ELMO, ELMO1 (T625) and ELMO2 (T615) (d), retroviral combinations of WT GRK6 and a DN form of GULP, GULP (PTB), as indicated (e). The retrovirus-infected NIH3T3 cells were subjected to the engulfment assay. Percentages of the phagocytes carrying the engulfed thymocytes were determined by FACS. All the phagocytosis experiments were done at least three times, and the average numbers are shown with s.e.m. n.s., not significant. ***P<0.001.
