Rac1 activity was measured by an affinity precipitation assay with Pak1 PBD agarose beads53 (link). BMDMs were lysed in 500 μl lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 4 mM EDTA, 4 mM EGTA, 10% glycerol, supplemented with protease inhibitor cocktail [Roche]). Ten percent of the whole cell lysate was set-aside for Rac1 loading control. To the remainder of the sample, 10 μg of Pak1 PBD beads were added, followed by a 45-minute incubation at 4°C. Beads were washed with lysis buffer and subsequently brought up in Laemmli sample buffer. Activated (GTP-bound) Rac1 was detected by immunoblot after affinity precipitation, and whole cell lysates were used as loading controls.
For active Rac1 immunofluorescence staining, human monocyctes or BMDMs were adhered, fixed and permeabilized, followed by primary staining with mouse anti-activated Rac1 (New East Biosciences) and secondary staining with Alexa Fluor 488 Goat Anti-Mouse IgM (Invitrogen). Corrected total cell fluorescence was quantified by ImageJ45 (link). Each individual data point represents an average CTCF of 4 fields of view with at least 20 cells per field of view.
For active Rac1 immunofluorescence staining, human monocyctes or BMDMs were adhered, fixed and permeabilized, followed by primary staining with mouse anti-activated Rac1 (New East Biosciences) and secondary staining with Alexa Fluor 488 Goat Anti-Mouse IgM (Invitrogen). Corrected total cell fluorescence was quantified by ImageJ45 (link). Each individual data point represents an average CTCF of 4 fields of view with at least 20 cells per field of view.