FIG. 8.

Nucleocytoplasmic shuttling of EB2 is not affected by the S3A mutation. F.EB2, and F.EB2S3A were expressed in HeLa cells. After 48 h, the HeLa cells were cocultivated overnight with NIH 3T3 cells. Prior to PEG-induced cell fusion, the cells were incubated for 2 h with cycloheximide. After fusion, the cells were further incubated for 2 h with cycloheximide. The cells were then immunostained using either the M2 anti-Flag MAb (panels e and h) or the anti-hnRNP-C (4F4) MAb (panel b) and an Alexa Fluor-conjugated secondary antibody. F-actin was labeled with Alexa Fluor 546 phalloidin to visualize the heterokaryons (panels c, f, and i). The cell nucleus was stained with Hoescht dye (panels a, d, and g). On the right sides of the panels the numbers indicate the numbers of heterokaryons with positive mouse cell nuclei immunostaining (indicating protein shuttling)/the total number of heterokaryons with positive HeLa nuclei immunostaining. IF, immunofluorescence.