Activation of mitotic checkpoint pathways in SUMO ligase-deficient cells. A, analysis of the activity of the replication stress checkpoint pathway. Wild-type (MMS21) or mms21Δsl cells having HA-tagged Mrc1 were subjected to HU-induced replication stress for varying time periods up to 3.5 h (left panels), and total lysates were analyzed by SDS-PAGE followed by Western blotting using anti-HA antibody (left panels). Modification of Mrc1 was observed in wild-type (top left panel) as well as mutant (bottom left panel) cell extracts. Cells were released from HU-induced arrest into YAPD medium lacking HU for varying time periods and analyzed for persistence of modification of Mrc1 (right panels), which was preferentially observed in the case of mutant cells. Mrc1 indicates the location of unmodified form; the arrow indicates the location of replication stress-induced modified forms. Similarly, wild-type (MMS21) or mms21Δsl cells having HA-tagged Rad53 were analyzed to detect the formation of HU-induced modified forms of Rad53 (position indicated by arrows). A, asynchronous cells prior to addition of HU. The lower panels are the corresponding regions from the Ponceau S-stained Western blots. B, activation of the spindle assembly checkpoint pathway in replication-stressed cells that were released into nocodazole (30 μg/ml)-containing medium for varying time intervals. Total lysates from wild-type (left) or mms21Δsl (right) cells were analyzed by SDS-PAGE followed by Western blotting using anti-Mad1 antibody (top panels). A, asynchronous cells prior to addition of HU; HU, cells treated with 150 mm HU for 3.5 h; the arrow indicates the position of the modified form of Mad1 that was observed after treatment with nocodazole. The lower panels are the corresponding regions from the Ponceau S-stained Western blots. Histograms show the results of FACS analysis, confirming mitotic arrest in nocodazole-treated wild-type (MMS21; left) and mms21Δsl (right) cell populations that were treated with HU for 3.5 h and released into nocodazole-containing medium (from bottom to top: asynchronous, 150 mm HU for 3.5 h, and released into nocodazole (30 μg/ml)-containing medium for 0.5, 1, 1.5, 2, 3, and 4 h).