Regulators and Effectors of Arf GTPases in Neutrophils

Figure 2

Expression of GIT2 in immune cells. RAW264 macrophages (1.5 × 10⁷ cells), human PMNs (3 × 10⁷), human lymphocytes/monocytes (LM, 3 × 10⁷), and dimethyl sulfoxide-differentiated HL-60 cells (3 × 10⁷) were mixed with an equal volume of boiling denaturing buffer and cell lysates were processed essentially as described by Marcil et al. [19]. The supernatants were then filtered through Sephadex G-10 columns to remove the denaturing agents and 0.1% Nonidet P-40, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 5 μL of bovine serum albumin (0.01% w/v) were added to the eluates. Samples were precleared with protein A-Sepharose and subsequently used for overnight immunoprecipitation with the polyclonal GIT2 antibodies 10 and 11 (5 μL). The beads were washed three times with ice-cold nondenaturing lysis buffer containing 1% Nonidet P-40 and boiled for 7 min at 100°C in 2x Laemmli’s sample buffer as described previously [19]. Immunoprecipitated proteins were electrophoresed on 10% SDS-PAGE and proteins were transferred to Immobilon PVDF membrane (Millipore Corp., Bedford, MA, USA). Membranes were incubated with the p95PKL/GIT2 antibody (P94020; 1 : 1500) from Bection Dickinson (Mississauga, ON, Canada) and exposed to peroxidase-conjugated anti-rabbit IgG (1 : 20,000) for 1 h at 37°C. The membranes were covered with ECL+ detection reagents. Images were obtained by exposing Kodak X-Omat film to membranes for 20 sec (upper panel) and 5 min (lower panel).