Figure 6
P2X7 and CD44 colocalize and closely interact in wild-type CHO-K1 cells. (a) Syndecan 1, (b) syndecan 2, (c) glypican 6 and (d) CD44 mRNA constitutive expression in CHO-K1 (black columns) and CHO-745 (white columns) cells, normalized over hypoxanthine–guanine phosphoribosyltransferase mRNA levels, and expressed as fold increases over controls (CTR, n=4). The data are represented as the means±S.E.M. (N=6). *P<0.05. (e) Immunofluorescence labeling with anti-P2X7 (green, first column) and anti-CD44 (red, second column) of CHO-K1 and CHO-745 cells. The nuclei were stained with DAPI (blue). Merge, third column. Scale bars, 20 μm. (f) P2X7 immunoprecipitation of whole-CHO-K1 cell lysate. Aliquots (500 μg) of cell lysates were incubated with polyclonal anti-P2X7 antibody overnight at 4 °C, followed by the addition of 20 μl of protein A/G PLUS-Agarose for 4 h at 4 °C. The proteins were resolved through 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The immunoprecipitated proteins were detected using anti-P2X7 and anti-CD44 antibodies, followed by incubation with the respective secondary antibodies conjugated to horseradish peroxidase. The bands were revealed using the SuperSignal West Pico Chemiluminescent Substrate.
