Ab128919

The Myc-Cdc5L and EGFP-securin plasmids were constructed for immunoprecipitation (IP). According to the previous description, the plasmids were transfected into 293T (Li et al., 2019 (link)). After 48 h, we collected the cells with 400-μl IP lysis buffer (Thermo Fisher Scientific, 88804) with the cocktail of protease and phosphatase inhibitors, followed by incubation on ice for 20 min. Next, we centrifuged the lysates at 13,400 rpm for 10 min at 4°C, and 400 μl of the supernatant was incubated with 1-μg primary antibody (GFP: abcam, ab290 or Myc: sigma, M4439) and 20-μl Magnetic Beads (Thermo Fisher Scientific, 88804) at 4°C overnight with rotation. The next morning, after washing five times, 40-μl 1 × sodium dodecyl sulfate loading buffer was added to the immunoprecipitate, and Western blotting was prepared. Western blot experiment was performed as described previously (Zhang et al., 2021 (link)). The anti-β-actin antibody (ZSGB-BIO, TA-09,1:1,000), rabbit anti-Cdc5L antibody (Thermo, 702831, 1:500), mouse monoclonal anti-Myc antibody (Sigma, M4439, 1:3,000), mouse monoclonal anti-Cyclin B1 antibody (Abcam, ab72, 1:1,000), and anti-SMC3 antibody (Abcam, ab128919, 1:1,000) were used for Western blotting.

The zona pellucida of the oocyte was removed by treatment with Acid Tyrode’s solution (Sigma-Aldrich) at room temperature. Then, the oocytes were placed onto glass slides and fixed in a solution described by Li et al. (2019) (link). The samples were placed in a humidifying box at room temperature for several hours, and then 2% bovine serum albumin was used for blocking at room temperature. The oocytes were then incubated with an anti-Bub3 antibody (1:200; ab133699, Abcam) and an anti-SMC3 antibody (1:20; ab128919, Abcam). After washing three times with washing buffer, the oocytes were incubated with the secondary antibody at room temperature for 1 h. Finally, 1 μg/ml DAPI was used to visualize the chromosomes.