Figure 1.

MORC2 is modified by SUMO1 and SUMO2/3. (A) Ectopically expressed Flag-MORC2 in HEK293T cells were pulled down with anti-Flag beads and analyzed by immunoblotting with indicated antibodies. (B) Endogenous MORC2 in MCF-7, T47D, and HEK293T cells were pulled down with an antibody against MORC2 and detected by immunoblotting with indicated antibodies. (C) Cells were treated with increasing doses of ML-792 (0, 0.01, 0.1 and 1 μM). Flag-MORC2 were pulled down with anti-Flag beads and analyzed by immunoblotting with antibodies against SUMO1 and SUMO2/3. (D) Endogenous MORC2 in MCF-7 and T47D cells was pulled down with an anti-MORC2 antibody and immunoblotted with indicated antibodies after treatment with increasing doses of ML-792 (0, 0.01, 0.1 and 1 μM). (E-F) HEK293T(E), MCF-7, and T47D cells (F) were fixed with paraformaldehyde solution and subjected to immunofluorescent staining with the indicated antibodies. Nuclei were counterstained with DAPI. Scale bar: 2.5 μm. (H-I) Total cellular lysates were subjected to IP assays with an anti-MORC2 (H) or anti-UBC9 (I) antibody, followed by immunoblotting using the indicated antibodies. (G) HEK293T cells were transfected with Flag-MORC2 and HA-UBC9 alone or in combination. Total cellular lysates were subjected to IP assays with anti-HA- or anti-Flag beads, followed by immunoblotting with the indicated antibodies. (J) HEK293T cells were transfected with Flag-MORC2, HA-UBC9 together with GFP-SUMO1, GFP-SUMO2 or GFP-SUMO3, respectively. Cellular lysates were pulled down with anti-Flag beads and then analyzed by immunoblotting. (K) MCF-7 cells were transiently transfected with or without HA-UBC9. Cellular lysates were used to IP assays with an anti-MORC2 antibody or control IgG, followed by immunoblotting with the indicated antibodies. (L) Endogenous UBC9 were knocked out in MCF-7 cells using two sgRNAs by the CRISPR/Cas9 system. Immunoprecipitated MORC2 were subjected to SUMOylation analysis.