Journal: eLife

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.7554/eLife.96810

Figure Lengend Snippet: ( A ) Representative bright-field pictures of wildtype (WT) and S100a9 -/- neutrophils spreading over E-selectin, ICAM-1, and CXCL1 coated glass capillaries (scale bars = 10 μm). Analysis of cell shape parameters ( B ) area, perimeter, ( C ) circularity (4π * [area/perimeter]) and solidity (area/convex area) over time (mean + SEM, n = 103 [WT] and 96 [ S100a9 -/- ] neutrophils of 4 mice per group, unpaired Student’s t-test). ( D ) Rose plot diagrams representative of migratory crawling trajectories of WT and S100a9 -/- neutrophils in flow chambers coated with E-selectin, ICAM-1, and CXCL1 under flow (2 dyne cm –2 ). Analysis of ( E ) crawling distance, ( F ) directionality of migration, and ( G ) crawling velocity of WT and S100a9 -/- neutrophils (mean + SEM, n = 5 mice per group, 113 [WT] and 109 [ S100a9 -/- ] cells, paired Student’s t-test). Western blot analysis of ICAM-1-induced ( H ) Pyk2 and ( I ) paxillin phosphorylation of WT and S100a9 -/- neutrophils upon CXCL1 stimulation (10 nM) (mean + SEM, representative western blot of n ≥ 4 mice per group, two-way analysis of variance (ANOVA), Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001. Figure 3—source data 1. Pyk2 and paxillin western blot data for . Original membranes corresponding to . paxillin, p-paxillin, Pyk2, and p-Pyk2 membranes are depicted and representative blots were then cropped and edited. Rectangle boxes indicate the representative bands used in the figure. Lowest membranes were cut before the staining to save staining solution and fit more membranes at the same time. GAPDH was always employed as an internal control. Chamaleon Duo Pre-stained Protein Ladder was used as molecular weight marker. Figure 3—source data 2. Pyk2 and paxillin western blot data for . Original membranes corresponding to . Paxillin, p-paxillin, Pyk2, and p-Pyk2 original membranes.

Article Snippet: After subsequent blocking (LI-COR blocking solution), membranes were incubated with the following antibodies for later detection and analysis using the Odyssey CLx Imaging System and Image Studio software: rabbit α-mouse phospho-paxillin (Tyr118) or rabbit α-mouse paxillin and rabbit α-mouse phospho-Pyk2 (Tyr402) or rabbit α-mouse Pyk2 (all Cell Signaling).

Techniques: Migration, Western Blot, Phospho-proteomics, Comparison, Staining, Control, Molecular Weight, Marker

Journal: eLife

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.7554/eLife.96810

Figure Lengend Snippet: ( A ) Representative bright-field pictures of wildtype (WT) and S100a9 -/- neutrophils spreading over E-selectin, ICAM-1, and CXCL1 coated glass capillaries (scale bars = 10 μm). Analysis of cell shape parameters ( B ) area, perimeter, ( C ) circularity (4π * [area/perimeter]) and solidity (area/convex area) over time (mean + SEM, n = 103 [WT] and 96 [ S100a9 -/- ] neutrophils of 4 mice per group, unpaired Student’s t-test). ( D ) Rose plot diagrams representative of migratory crawling trajectories of WT and S100a9 -/- neutrophils in flow chambers coated with E-selectin, ICAM-1, and CXCL1 under flow (2 dyne cm –2 ). Analysis of ( E ) crawling distance, ( F ) directionality of migration, and ( G ) crawling velocity of WT and S100a9 -/- neutrophils (mean + SEM, n = 5 mice per group, 113 [WT] and 109 [ S100a9 -/- ] cells, paired Student’s t-test). Western blot analysis of ICAM-1-induced ( H ) Pyk2 and ( I ) paxillin phosphorylation of WT and S100a9 -/- neutrophils upon CXCL1 stimulation (10 nM) (mean + SEM, representative western blot of n ≥ 4 mice per group, two-way analysis of variance (ANOVA), Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001. Figure 3—source data 1. Pyk2 and paxillin western blot data for . Original membranes corresponding to . paxillin, p-paxillin, Pyk2, and p-Pyk2 membranes are depicted and representative blots were then cropped and edited. Rectangle boxes indicate the representative bands used in the figure. Lowest membranes were cut before the staining to save staining solution and fit more membranes at the same time. GAPDH was always employed as an internal control. Chamaleon Duo Pre-stained Protein Ladder was used as molecular weight marker. Figure 3—source data 2. Pyk2 and paxillin western blot data for . Original membranes corresponding to . Paxillin, p-paxillin, Pyk2, and p-Pyk2 original membranes.

Article Snippet: After subsequent blocking (LI-COR blocking solution), membranes were incubated with the following antibodies for later detection and analysis using the Odyssey CLx Imaging System and Image Studio software: rabbit α-mouse phospho-paxillin (Tyr118) or rabbit α-mouse paxillin and rabbit α-mouse phospho-Pyk2 (Tyr402) or rabbit α-mouse Pyk2 (all Cell Signaling).

Techniques: Migration, Western Blot, Phospho-proteomics, Comparison, Staining, Control, Molecular Weight, Marker