Research Paper Volume 10, Issue 3 pp 402—424

Preferential Ty1 retromobility in mother cells and nonquiescent stationary phase cells is associated with increased concentrations of total Gag or processed Gag and is inhibited by exposure to a high concentration of calcium

Figure 7. Mother cells have increased levels of Gag. (A) Western blot of protein extracts from cells grown in YPD to late exponential phase at the indicated temperatures. Upper panel shows the blot probed with Gag antibody and the lower panel shows the blot stripped and reprobed with beta-actin antibody. Arrows indicate the migration of the alternative forms of Gag, and the number to the left shows the migration of a 50 kD protein size standard. (B) Western blot for Gag protein present in extracts of sorted mother (M) and daughter (D) cells grown in YPD without or with 100 mM calcium chloride, as described for panel A. An extract from an spt3∆ strain was used as a control for low Gag expression. (C) Quantification of the Gag signal normalized to beta-actin from Western blots of mother and daughter cell extracts grown with or without 100 mM calcium chloride. Data are mean and standard deviation values for three trials. Horizontal bars indicate comparisons between mothers and daughters. Asterisks over individual columns indicate comparisons to mock treatment. Symbols for statistical significance are as for Fig. 2. (D-E) Westerns blots of extracts prepared from mother (M), daughter (D), nonquiescent (NQ), quiescent (Q), and exponential phase (Exp) cells expressing a Gag-GFP fusion protein, or a control strain lacking the fusion protein (no GFP). Upper two blot panels in each case were probed with a GFP antibody, with the top image being overexposed to show p22-GFP (arrow). Middle image shows a shorter exposure for Gag-GFP, and bottom image shows the blot stripped and reprobed with beta-actin antibody. Some extracts were prepared from cells grown in 100 mM calcium chloride for panel E, as indicated. Migration of 85 and 50 kD size standards is indicated to the left for the top image.