Expression of FLNa and FLNb in immature and mature DCs. (A) Expression of FLNa, FLNb, and α-actinin 1 was analyzed by western blot using 15-μg aliquots of whole-cell extracts of untreated (-) and LPS-treated BMDCs as indicated. (B) Expression of FLNa was assessed by immunofluorescence in immature BMDCs cultured without (-) or with LPS for 24 hours. Cells were harvested, centrifuged onto poly-l-lysine–coated glass coverslips, fixed, stained for FLNa and phalloidin, and imaged (left panel). Dot plots show the overall distribution of relative FLNa fluorescence intensities, and lines show the median values (right panel). Scale bar represents 20 μm. (C) Expression of FLNa was assessed by flow cytometry in immature BMDCs cultured without (-) or with LPS for 24 hours. After fixation and permeabilization, cells were stained with anti-FLNa and alexa Fluor 488-conjugated anti-rabbit antibodies. White and gray areas show representative staining profiles of immature and LPS-treated BMDCs, respectively (left panel). The relative quantification of FLNa expression in untreated and LPS-treated BMDCs is shown as means and standard deviation (SD) of 3 independent experiments (right panel). (D) Relative expression of FLNa mRNAs in untreated (-) and LPS-treated BMDCs assessed by quantitative real-time RT-PCR. Levels were normalized to Arbp. The data show means and SEM of 3 independent experiments. ***P < .001.