Fcγ Receptor induced phagocytic activity is decreased following the treatment by PMA in J774A.1 cell line. (A) For PMA treatment, cells were pre incubated with PMA (0.2 μg/ml or 0.4 μg/ml) for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X for 15 min. followed by PMA (0.4 μg/ml) treatment. To initiate phagocytosis, media was replaced by 1ml IgG coated sheep RBCs and incubated for 30 min. at 37oC. (B) Effect of over expression of SHP-1 on CBL dephosphorylation.. J774A.1 cells were infected either with empty vector recombinant virus (pSC 65) or wild type SHP-1 (MOI 2 pfu/cell for overnight) or with C2 mutant of SHP-1. P and NS represents pre-immune and non-stimulation respectively, non infected serve as control. Except P and NS conditions, all cells were stimulated with sensitized sheep RBCs for 10 min. Immunoblots were reprobed with anti-CBL, anti-SHP-1 and anti-CRKL antibody. Lower panel shows the Western blot analysis of over expression of SHP-1 and C2 mutant in J774A.1 cells. (C) J774A-1 cells were infected with pSC 65or recombinant vaccinia for the expression of C2 mutant or SHP-1 at an MOI of 2 pfu / cell for 4 hours at 37oC as described in Methods. Western blot analysis confirms equivalent level of over expression of SHP-1 and C2 mutant in J774-1 cells. (D). Peritoneal macrophages isolated from C57BL/6 mice were pre incubated with PMA for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X (above mentioned conc.) for 15 min. followed by PMA (0.4 μg/ml) treatment and phagocytosis was performed as described above. Each bar represents mean ± SD, n=3. *P <0.05, **P <0.01 and ***P <0.001 vs. control (t-test) All these experiments were performed three times.
Joshi et al. BMC Immunology 2014 15:18 doi:10.1186/1471-2172-15-18