Fig. 4.
Arx1 fails to recycle in Δjjj1 cells. (A) Approximately equal numbers of cells of the indicated genotypes were diluted 10-fold, spotted onto rich media, and incubated at 23°C. (B) Localization of Arx1-GFP was monitored by fluorescence microscopy in the indicated strains. All cells were grown at 25°C in rich media, except Δjjj1 carrying pRS316 jjj1HPD→AAA (jjj1HPD→AAA), which was cultured in minimal media lacking uracil. (C) Lysates from 25°C-grown cells containing chromosomally integrated ARX1-HA were centrifuged through 7–47% sucrose gradients and the OD254 monitored (Upper). Fractions were analyzed for the presence of Arx1-HA and Rpl8 by immunoblot analysis (Lower). (D) Extracts from WT or Δjjj1 cells containing genomically integrated Arx1-HA were incubated with anti-HA antibody and protein A beads. Precipitated proteins were eluted from protein A beads in sample buffer and separated by SDS/PAGE. Total protein extract (T) as well as immunoprecipitation from an untagged strain (C) were included as controls. Immunoblotting was performed by using antibodies specific for the HA or Rpl8.