The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects -- Data Supplement - JCB--Chiroli et al. -- The Journal of Cell Biology

[HELP with High Resolution Image Viewing]

Figure S3. Lack of Cdc55 allows anaphase in the absence of Mcd1 cleavage in CLA4t-overexpressing cells. 4X GAL1-CLA4t (ySP5480) and 4X GAL1-CLA4t cdc55Δ (ySP5578) cells expressing HA-tagged Mcd1 (Mcd1-HA3) growing at 30°C in YEPR were arrested in G1 by α factor and released in YEPRG at 25°C (time 0). Samples were collected at the indicated times for determination of DNA contents (A), budding and nuclear division (B), Western blot analysis of TCA protein extracts (C and D), and chromosome spreads (E). Total extracts were immunoblotted with anti-HA antibodies to detect Mcd1-HA3. The same blot shown in the top panel for Mcd1-HA3 was overexposed to visualize Mcd1 cleavage products. The same protein extracts as in C were loaded in parallel with a TCA protein extract from wild-type cells treated with nocodazole for 3 h to detect phosphorylation of Mcd1-HA3 (D). Note that in nocodazole Cdc5-dependent Mcd1 phosphorylation makes it run as a doublet. Chromosome spreads (E) were immunostained with anti-HA antibodies (Mcd1-HA3) and mounted with DAPI to stain DNA. Arrows indicate anaphase nuclei.