Av40203

To analyse ANP32A expression in gene-edited clonal lines (Supplementary Fig. 3 and Supplementary Fig. 17k), at least 150,000 cells were lysed in 50 µl of 1X RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology) containing protease and phosphatase inhibitor (Halt, Thermo Scientific 78440) according to the manufacturer’s instruction. To analyse ANP32A expression in AKO embryos (Supplementary Fig. 14g), approximately 2 mg of embryonic tissue was lysed in 100 µl of 1X RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology) containing protease and phosphatase inhibitor (Halt, Thermo Scientific 78440) according to the manufacturers’ instruction. Denaturing electrophoresis and western blotting were performed using the NuPAGE electrophoresis system (Invitrogen) following the manufacturer’s protocol. Immunoblotting was performed using the following primary antibodies; rabbit anti-ANP32A (Sigma-Aldrich AV40203; 1/1000 dilution) and mouse anti-ɣ-tubulin (Sigma-Aldrich TS6557; 1/1000 dilution). The following secondary antibodies were used: goat anti-mouse IRDye 800CW (LI-COR 925-32211; 1/10,000 dilution) and goat anti-rabbit IRDye 680RD (LI-COR 925-68070; 1/10,000 dilution). Protein bands were visualised through fluorescence using the Odyssey Imaging System (LI-COR) according to the manufacturer’s instruction.

huANP32A, huANP32B, chANP32A and their truncated versions were probed with rabbit anti-huANP32A (ab155148, Abcam), rabbit anti-huANP32B (ab200836, Abcam), and rabbit anti-chANP32A (AV40203, Sigma) antibodies. Beta-actin was probed with a mouse anti-beta-actin antibody (sc-47778, SCBT). Goat anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase (HRP) were used as secondary antibodies. Detection was carried out using Amersham ECL Western blotting detection reagents (GE Healthcare).