Fig. 2
From: MAPK-dependent control of mitotic progression in S. pombe
Pmk1 deletion leads to increased levels of Cdc20Slp1, but does not impair MCC formation or the association of MCC to APC/C. A Top. Microscopy images from asynchronously growing cells expressing GFP tagged version of the indicated SAC components. A kinetochore marker (Cnp1-Tomato) was used to prove proper kinetochore recruitment of SAC components (not shown). Graphs represent the average maximum signal intensity at kinetochore of the indicated markers. Error bars indicate the SD of n=40 cells for each condition from two independent experiments; n.s.= not statistically significant (p > 0.05). Bottom. Western blotting showing total protein levels of Mad1, Mad2, Mad3 Bub3, and Bub1 in the wild type and pmk1Δ strains. Cdc2 was used as loading control. B Co-IP experiment pulling down Slp1-HA and detecting Mad3-GFP. Cells were harvested from a mitotic block. Untagged Slp1 was used as a negative control, whereas Mad3- Cdc20Slp1 interaction in a wild type was used as a positive control. IP: Immunoprecipitation, IB: Immunoblotting. C Co-IP experiment pulling down APC/C component Apc4. Untagged Apc4 was used as a negative control, whereas Mad3-Apc4 interaction in a wild type was used as a positive control. D Western blot of total Cdc20Slp1 protein levels in asynchronously growing wild type, pmk1Δ and pmk1-KD cells under the same conditions. Cdc2 was used as loading control. Quantification of Cdc20Slp1 total levels relative to loading control is shown on the bar graph. Asterisks indicate statistical significance (p<0.01). E Cdc20Slp1 mRNA analysis was done by Q-PCR from asynchronously growing wild type and pmk1Δ cells. CT values were relativized with actin, which was used as a control, and to the wild type. Graph represents data from 3 independent repeats with 6 technical replicas; n.s. means differences are not statistically significant (p > 0.05). A–E Images show a representative example of two independent experiments.  F Cells from strains cdc25-22 and pmk1Δ cdc25-22 were grown to O.D.= 0.3 at 25°C, shifted to 36°C for 3.5 h, and then released to permissive temperature (25°C). Samples were taken at the indicated time points. Slp1-HA was detected along with activated Pmk1 with anti-phosho-p44/42 antibody. Anti-tyrosine-Cdc2 antibody was used to follow G2/M transition. Cdc2 was used as a loading control. Percentage of septated cells and Cdc20Slp1 levels at each time point is indicated on the graph. G Western blot of mitotic Cdc20Slp1 levels in nda3-KM311 and pmk1Δ nda3-KM311 cells. Samples were taken at the indicated time points after the shift to 18°C. Cdc2 was used as loading control. Percentage of septated cells and Cdc20Slp1 levels at each time point are indicated on the graph. F, G Images show a representative example of three independent experiments