ABL1 kinase activity is essential for PXN Y118 phosphorylation in HDMECs in vitro. (A–C) To examine if ABL1 kinase activity is essential for PXN phosphorylation, we performed immunofluorescence labeling (A and B) and immunoblotting (C) of HDMECs treated with vehicle or Imatinib 30 min before and during plating on FN for the indicated times. In A, pPXN Y118 (green) is shown together with phalloidin (red) and DAPI (blue) on the left and as single channel in grayscale on the right. Bars, 20 µm. pPXN pixel intensity was quantified in B and expressed as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SD of 4 independent experiments). *, P < 0.05, Student’s t test. (C) Immunoblotting confirmed that Imatinib treatment reduced pPXN Y118 levels. Total ERK1/2 levels were used as a loading control. (D) To examine if endogenous NRP1 and ABL1 form a complex in ECs, we performed coimmunoprecipitation of endogenous proteins from lysates of HDMECs, treated with vehicle or 10 µM Imatinib for 30 min, detached, and plated on FN for the indicated times in the presence of Imatinib. Immunoprecipitation using ABL1 antibody was followed by immunoblotting performed for NRP1 and ABL1.