Supplemental Figure

This article contains the following supporting material:

• Supplemental Figure 1 - Figure S1. Inhibition of dye uptake induced by fluid flow shear stress by an antibody raised against the second extracellular loop (E2) of Cx43. (A) Immunoblots of membrane preparation isolated from MLO-Y4 cells were labeled with 1:300 dilution of affinity purified anti-Cx43(E2) blocking antibody (Ab) and preimmune antibody (Pre). This antibody recognized a protein band at 43 kDa by Western blots (Ab), while no specific band was detected by preimmune antibody (Pre). (B) MLO-Y4 cells were first labeled with 1:300 dilution of affinity purified anti-Cx43(E2) blocking antibody (Ab) or preimmune antibody (Pre) and then 1:500 dilution of fluorescein-conjugated anti-rabbit IgG. Immunostaining showed the labeling of Cx43 with Cx43(E2) antibody (Ab), but no labeling with preimmune antibody (Pre). (C) The number of cells with dye uptake with and without fluid flow, preimmune (Pre) or anti-Cx43(E2) antibody (Ab) were counted and quantified. As compared to untreated control (C), fluid flow shear stress (FF) stimulated the uptake of the dye in MLO-Y4 cells and this increase is blocked by anti-Cx43(E2) blocking antibody (30 ?g/ml) (Ab), but not by preimmune antibody (Pre). C, C + Ab, C + Pre, or FF + Ab versus FF or FF + Pre: ***, ? < 0.001. (D) Scrape loading dye transfer analysis did not show any difference in cells treated with preimmune (Pre) or anti-Cx43(E2) antibody (Ab). Together, these results indicate that fluid flow-induced dye uptake was mediated by Cx43-hemichannels.

Prepared by: Journals Production Manager