Figure 2. Premature degradation of APC/C... | Rockefeller University Press

Figure 2.

$Figure 2. Premature degradation of APC/C substrates in cells depleted of MAD2L2. (A) Silencing of MAD2L2 results in a lower mitotic index in nocodazole. The percentage of mitotic U2OS cells was calculated by flow cytometry monitoring histone H3 phospho-serine 10 in cells treated with nocodazole for 16 h. Error bars = 1 SD. (B) APC/C substrate degradation in control siRNA-treated U2OS cells. (C) APC/C substrate degradation in siMAD2L2-treated cells. The asterisk indicates remnant Cyclin B1 signal in the AURKA blot. (D) APC/C substrate degradation in cells complemented with siRNA-resistant hMAD2L2-myc. (E) Summary of substrate degradation in control and siMAD2L2-treated cells in the 180 min after nocodazole release. Substrate levels at each time point are normalized to actin and then shown as a fraction of the level of the substrate at t = 0 in control siRNA-treated cells. The curve fit is an exponential decay. The t1/2 and fitting statistics are presented in Table 2. Error bars = SEM. Control siRNA: solid black circle/solid black line, n = 5. siMAD2L2: solid red triangles/solid red line, n = 3. siMAD2L2 complemented with hMAD2L2-myc: open circles/dashed black line, n = 4. siREV3: solid blue square/solid blue line, n = 3. “n” represents the number of independent experiments. The additional blots that contribute to this analysis are shown in Fig. S1 (control, siMAD2L2, and complemented) and Fig. S4 (siREV3).$

Premature degradation of APC/C substrates in cells depleted of MAD2L2. (A) Silencing of MAD2L2 results in a lower mitotic index in nocodazole. The percentage of mitotic U2OS cells was calculated by flow cytometry monitoring histone H3 phospho-serine 10 in cells treated with nocodazole for 16 h. Error bars = 1 SD. (B) APC/C substrate degradation in control siRNA-treated U2OS cells. (C) APC/C substrate degradation in siMAD2L2-treated cells. The asterisk indicates remnant Cyclin B1 signal in the AURKA blot. (D) APC/C substrate degradation in cells complemented with siRNA-resistant hMAD2L2-myc. (E) Summary of substrate degradation in control and siMAD2L2-treated cells in the 180 min after nocodazole release. Substrate levels at each time point are normalized to actin and then shown as a fraction of the level of the substrate at t = 0 in control siRNA-treated cells. The curve fit is an exponential decay. The t_1/2 and fitting statistics are presented in Table 2. Error bars = SEM. Control siRNA: solid black circle/solid black line, n = 5. siMAD2L2: solid red triangles/solid red line, n = 3. siMAD2L2 complemented with hMAD2L2-myc: open circles/dashed black line, n = 4. siREV3: solid blue square/solid blue line, n = 3. “n” represents the number of independent experiments. The additional blots that contribute to this analysis are shown in Fig. S1 (control, siMAD2L2, and complemented) and Fig. S4 (siREV3).