Figure 6. PP2A Cdc55 -mediated... | Rockefeller University Press

Figure 6.

$Figure 6. PP2ACdc55-mediated dephosphorylation of APC subunits is essential for spindle checkpoint activity. (A) CDC16-myc6 (yAC1936), CDC16-myc6 cdc55Δ (yAC1994), CDC27-myc9 (yAC1863), and CDC27-myc9 cdc55Δ (yAC1888) cells were grown in YEPD at 30°C, arrested in G1 phase, and released in nocodazole. Samples were taken during the G1 arrest and after 2 and 3 h for Western blotting analysis of Cdc16 (left) and Cdc27 (right) using Phos-tag reagent. The same samples were loaded on standard 10% acrylamide gels to assess the total levels of Pgk1, Cdc16, and Cdc27. In the bottom panels, the fraction of phospho-specific shifts on P-tag gels was normalized to the total amount of protein. Error bars refer to standard errors of three independent experiments. (B–E) GAL1-MAD2 (3X) PDS1-MYC18 cdc55Δ (yAC1823), GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A (yAC1675), and GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A cdc55Δ (yAC1952) cells were arrested in G1 with α-factor and released in galactose. Samples were collected for IF (B) and Western blotting analysis (C–E). The data shown in B are from a single representative experiment out of three repeats. For the experiment shown, n = 100.$

PP2A^Cdc55-mediated dephosphorylation of APC subunits is essential for spindle checkpoint activity. (A) CDC16-myc6 (yAC1936), CDC16-myc6 cdc55Δ (yAC1994), CDC27-myc9 (yAC1863), and CDC27-myc9 cdc55Δ (yAC1888) cells were grown in YEPD at 30°C, arrested in G1 phase, and released in nocodazole. Samples were taken during the G1 arrest and after 2 and 3 h for Western blotting analysis of Cdc16 (left) and Cdc27 (right) using Phos-tag reagent. The same samples were loaded on standard 10% acrylamide gels to assess the total levels of Pgk1, Cdc16, and Cdc27. In the bottom panels, the fraction of phospho-specific shifts on P-tag gels was normalized to the total amount of protein. Error bars refer to standard errors of three independent experiments. (B–E) GAL1-MAD2 (3X) PDS1-MYC18 cdc55Δ (yAC1823), GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A (yAC1675), and GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A cdc55Δ (yAC1952) cells were arrested in G1 with α-factor and released in galactose. Samples were collected for IF (B) and Western blotting analysis (C–E). The data shown in B are from a single representative experiment out of three repeats. For the experiment shown, n = 100.