NME/β3 integrin interaction occurs at α-adaptin–marked CCPs and does not need β1 integrin in osteoblasts. (A) PLA signal using NME antibody in combination with ICAP-1 antibody (4d1d6) or ICAP-1 antibody (9b10) or β3 integrin antibody or β1 integrin antibody in WT osteoblasts. Immunolabeling of α-adaptin was performed after PLA. Insets are higher magnification of boxed regions. Insets show PLA signals (NME/ICAP-1 or NME/β1 integrin or NME/β3 integrin) colocalizing with α-adaptin-positive CCPs (arrowheads). PLA signal indicates a close proximity, possibly in situ interaction of NME with ICAP-1, β1, and β3 integrins at CCPs. Scale bar, 10 μm. Inset scale bar, 5 μm. (B and C) Representative images of PLAs (B) and PLA assay quantification (C) of the number of PLA spots per cell performed with antibodies against NME and β3 integrin. Red dots denote regions of signal amplification consistent with NME/β3 integrin proximity in osteoblast cells deficient or not in β1 integrin. PLA performed on ICAP-1 and β1 integrin deficient cell line is used as control. Scale bar, 20 μm. Error bars represent SD. N = 15 cells/condition from three independent experiments. ns, adjusted P value > 0.05.