Fig 6.

USP1 instability in CAPNS1-depleted cells can be rescued by cdh1 depletion or Cdk5/p25 overexpression. (a and b) U2OS cells were transfected with the indicated siRNA, and 48 h later, the cells were incubated or not with mimosine (a) to induce G₁/S arrest or with nocodazole to achieve arrest in M phase (b). Sixteen hours later, cell lysates were collected and analyzed with the indicated antibodies. (c) U2OS cells were transfected with the indicated siRNAs, and 72 h later, the respective lysates were utilized for Western blots to monitor the USP1 level. (d and e) p35-overexpressing U2OS cells (d) or MCF10A cells (e) were transfected with the indicated siRNAs, and 48 h later, the cells were incubated with mimosine to induce G₁/S arrest. The cells were then irradiated with 30 J/m² UV light, and at the indicated time points, lysates were collected and analyzed by Western blotting with the indicated antibodies. (f and g) U2OS cells were transfected with the indicated plasmids and kept under selection for 2 weeks to increase the percentage of cells overexpressing p25 or p35. Afterwards, they were transfected with the indicated siRNAs, and 48 h later, the cells were left untreated (f) or were incubated with mimosine (g). Sixteen hours later, cell lysates were utilized for Western blots to monitor USP1 levels.