LPS stimulation of primary macrophages results in a decrease in macropinocytic activity and SNX5 levels. (A-D) Bone marrow-derived macrophages were grown for 7 days in M-CSF and then analysed as follows. (A) Cells were cultured in the presence of 100 μg/ml TR-conjugated dextran (10,000 MW) for 3 min. The cells were washed at 4°C and fixed in 4% PFA and stained with affinity-purified SNX5 antibodies followed by Alexa488-conjugated goat anti-rabbit IgG. Bar = 10 μm. (B) Macrophages were seeded at 1 × 107 cells/ml and incubated with 10 ng/ml LPS for up to 24 h, as indicated. Samples (15 μg protein) were separated by SDS-PAGE and probed by immunoblotting using the anti-SNX5 antibody. (C-D) Macrophages were either left untreated or treated with 10 ng/ml LPS for 24 h and then pulsed with 1 mg/ml Alexa 488-conjugated dextran for 3 minutes at 37°C and fixed in 4% PFA. The samples were then stained with Texas-red-X conjugated phalloidin. Images were captured using identical settings for -/+ LPS conditions and counted as described in Methods. At least 180 cells were analysed for each condition.