Immunofluorescence microscopy
Transfected HEK293 cells were fixed and stained as described previously . Primary antibodies anti-VWF (Abcam, England) and anti-protein disulfide isomerase (PDI; Life technologies, USA) were used to visualize intracellular VWF and endoplasmic reticulum (ER), respectively. Z-stack images were acquired with Zeiss Apotome.2 microscopy (Carl Zeiss, Germany). The quantitative co-localization investigation was done using Zeiss ZEN 2 (blue edition; Carl Zeiss, Germany). Analysis for each set of comparisons was performed on z-series of images of a minimum of 100 cells from two independent experiments (each in triplicate). The comparative degree of co-localization was calculated as mean Pearson’s correlation coefficients.