Figure 5.

ANGPTL4 interacts with integrins β1 and β5. Representative sensorgrams showing binding profiles between immobilized cANGPTL4 and S2-membrane extracts containing either control, integrin β1, β3, or β5 (A), or integrin β1 or β5 preincubated with either preimmune IgG (pre) or cognate anti-integrin antibody (B). Integrin β1 (C) or integrin β5 (D) after preblocked with either preimmune IgG or anti-cANGPTL4 antibody. Each sensorgram was corrected by subtracting a sensorgram obtained from a reference flow cell with no immobilized protein. Anti-cANGPTL4 antibodies against the immobilized cANGPTL4 determined the Rmax value to be 138.2 resonance units (RU). Five independent experiments were performed. Dose-dependent ANGPTL4 binding to immobilized (E) integrin α5β1 or (F) integrin αvβ5, which was specifically blocked by anti-cANGPTL4, as determined by ELISA. Detection of (G) the ANGPTL4-integrin β1 (left panel) and ANGPTL4-integrin β5 (right panel) complexes in K_CTRL and in day-5 ANGPTL4^+/+ wound biopsies using DUOlink PLA. PLA signals (red) and Hoechst dye for nuclei (blue). For K_CTRL, the cells were counterstained with Alexa 488-phallodin for actin stress fibers. The nuclear image was acquired in one z-plane using a LSM510 META confocal laser-scanning microscope (Carl Zeiss). Dotted white line represents epidermal-dermal junction. Negative control was performed without primary antibodies. Representative pictures from wound sections with epidermis (e), dermis (d), wound bed (wb), and K_CTRL from six independent experiments or sections from three mice are shown. Scale bar = 40 μm. H: Immunodetection of indicated proteins from anti-cANGPTL4 immunoprecipitates of ANGPTL4^+/+ and ANGPTL4^−/− wound biopsy homogenates. Total ERK from supernatant were used to verify equal loading.