Fig. 2.

Antibody IPS-K-4A2B8 recognizes a GSL in lipid rafts. Open, blue and red histograms represent controls, untreated and treated cell staining, respectively. (A, B) Resistance of IPS-K-4A2B8 antigen to solubilization with TX-100. Native (A) and TX-100 (B) extracted iPS122 or DU145 cells were stained with IPS-K-4A2B8 or CD44 antibody followed by PE-labeled secondary antibody. Note that both CD44 and IPS-K-4A2B8 antigen are resistant to solubilization with TX-100. (C) IPS-K-4A2B8 antigen colocalizes with the lipid raft marker CTB in DU145 cells. DU145 cells were preincubated with mAb IPS-K-4A2B8 and then with the lipid raft marker Alexa Fluor 555-CTB at 4°C as described in Methods. IPS-K-4A2B8 antigen and CTB strongly colocalize at the plasma membrane. (D and E) Resistance of IPS-K-4A2B8 antigen to proteinase K. iPS122 and DU145 cells were treated with proteinase K to strip off the membrane proteins. Untreated (D) or treated (E) cells fixed with 1% paraformaldehyde and stained with IPS-K-4A2B8 or CD44 antibody. Note that the reactivity of mAb IPS-K-4A2B8 is retained, whereas CD44 expression is completely lost after proteinase K digestion. Untreated (F) and PI-PLC-treated (G) iPS122 cells were stained with IPS-K-4A2B8 or tissue non-specific alkaline phosphatase antibody (clone: IPS-K-4A2B8). (H and I) MβCD treatment abrogates binding of IPS-K-4A2B8 antibody. PC3 cells were incubated with (I) and without (H) 5 mM MβCD at 37°C for 1 h. The cells were then stained with anti-CD44 or IPS-K-4A2B8 antibody. Note that the binding of mAb IPS-K-4A2B8 is completely lost after MβCD treatment.