Co-localization of MBP with galactolipids at the plasma membrane of OLN-93 cells and its microdomain association.

<p>OLN-P, OLN-G and OLN-GS cells were cultured on PLL and transiently transfected with 18.5-kDa MBP-eGFP. The experiments were performed 24 hours after transfection. <b>A,B</b>) Representative images of transfected cells stained for either cell surface GalC or sulfatide with O1 and O4 antibodies (red), respectively. Scale bar is 6 µm. Insets show higher power magnifications of which brightness-contrast and median filtering were performed by Image J. <b>C</b>) The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient Calculator (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101834#s2" target="_blank">Materials and Methods</a>) and displayed as a bar graph. Bars represent the mean+SEM of at least 15 cells. The statistical analysis was performed by GraphPad Prism 5 (one-way ANOVA followed by the Newman-Keuls posttest, * p<0.05). <b>D–F</b>) OptiPrep density gradient centrifugation analysis after CHAPS extraction. Caveolin-1 and Rho-GDI are a positive and negative marker for membrane microdomains (lipid rafts), respectively (<b>D</b>). 18.5-kDa MBP-eGFP expression was determined using an anti-MBP polyclonal antibody (<b>E</b>). Representative Western blots are shown. The intensities of the MBP-eGFP bands in <b>E</b> were quantified by Image J. The total protein expression was calculated by adding the intensity of all the fractions. The protein percentage of each fraction was then calculated by dividing the protein intensity present in that fraction by total protein expression. A bar graph of the distribution of MBP-eGFP is shown. The fraction percentage of (raft) fractions 3 and 4 are plotted in black and the fraction percentage of (non-raft) fractions 6 and 7 are shown as grey bars. Bars represent the mean+SEM. The statistical analysis was performed using GraphPad Prism 5 (n = 3, one-way ANOVA followed by the Newman-Keuls posttest, ** p<0.01).