Paxillin-deficient cells form podosomes with smaller actin cores and strongly reduced lifetime. (A) Kindlin-3, leupaxin, PTP-PEST, and talin expression in +/+ RAW cells and four different paxillin^−/− RAW cell clones analyzed by Western blotting. (B) IF stainings of +/+ and paxillin^−/− RAW cells for vinculin (green), paxillin (red), and actin (white/blue in merge). (C) Diameter of the podosome actin cores in +/+, paxillin^−/−, and leupaxin^−/− RAW cells. 10 actin cores in two regions of five to eight cells were measured in each experiment. n = 10/6/7. (D) IF stainings for vinculin (green), kindlin-3 (red), and actin (white/blue in merge) on +/+ and paxillin^−/− RAW cells. (E) IF stainings for talin (green), PTP-PEST (red) and actin (white/blue in merge) on +/+ and paxillin^−/− RAW cells. (F) Control RAW cells and different clones of paxillin^−/− RAW cells were transfected with LifeAct-GFP and imaged at a spinning disc microscope for 10 min with a 15-s time interval. The cumulative distribution of these measurements is shown. 10–30 podosomes were measured per cell. At least four cells were analyzed in each of six independent experiments. (G) Confocal images of talin (green), leupaxin (red), and actin (white/blue in merge) IF stainings of +/+ and paxillin^−/− RAW cells. (H) IF stainings shown in G were quantified by measuring fluorescence intensity. Values from WT cells were set to 1. In each independent experiment, five podosome regions in each of at least 10 cells were measured. n = 4. Scale bars, 10 µm. Dotted white lines mark cell borders.