Figure 3.

Analysis of cell growth and proteins produced by yeast cells harboring the UB-pol30 and pol30-UB gene fusions. (A) Cell growth by a colony size assay. Equal inoculations of HK578-10D pol30Δ::HIS3 cells harboring different plasmids as indicated were streaked onto YPD agar and incubated at 30°C for 48 h. Differences in growth rates are observed if the colony sizes vary noticeably between strains. (B) Analysis of PCNA protein from whole-cell lysates. POL30 deletion strain HK578-10D pol30Δ::HIS3 containing the plasmids as indicated was used to create whole-cell lysates. The anti-PCNA western blot reveals the PCNA derivatives contained within each strain. Non-specific cross-reacting bands indicated with asterisks demonstrate equal protein loading throughout the samples. (C) Analysis of affinity purified PCNA protein. Lysates made from HK578-10D pol30Δ::HIS3 derivatives that produce 7xHis-tagged PCNA were subjected to His-affinity chromatography. Western blots of the samples using anti-PCNA (left), anti-Ub (center), and anti-His₆ (right) antibodies are shown. The non-specific co-purifying bands detected (marked with asterisks) in the anti-His blot indicate that similar amounts of total protein were used for each sample.