Senp2

In situ hybridization was performed as described11 (link)30 (link)31 (link)32 (link). In brief, sections were incubated with the digoxygenin labeled RNA probes generated by in vitro transcription11 (link)31 (link), followed by recognition with an alkaline phosphatase conjugated anti-digoxygenin antibody, and visualization with BM-purple30 (link)31 (link)32 (link). Immunostaining of cells11 (link)33 (link) and tissue sections11 (link)34 (link)35 (link)36 (link) were performed by incubation with primary antibodies, followed by detection with fluorescence-conjugated or horseradish peroxidase-conjugated secondary antibodies. Images were taken using Zeiss Axio Observer microscope equipped with deconvolution analysis19 (link)31 (link)36 (link). Immunoblot was performed by isolation of protein extracts from E9.5 embryos using M-PER (Pierce) in the presence of protease inhibitor cocktail, followed by electrophoresis as described19 (link)30 (link)34 (link). Mouse monoclonal antibodies, Actin (Thermo Fisher), Mdm2 (Santa Cruz), p57^Kip2 (Thermo Fischer); rabbit polyclonal antibody, SENP2 (Abgent) were used as primary antibodies as indicated.

Tissues and cells were homogenized in whole-cell lysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 1.0% NP-40) containing a protease inhibitor cocktail and 20 mM N-ethylmaleimide to prevent SUMO de-conjugation. Immunoblotting was performed with primary antibodies against HOXA10 (1 : 1000; Santa Cruz, Santa Cruz, CA, USA), SUMO1 (1 : 1000; Abcam, Cambridge, CA, USA), SENP1 (1 : 1000; Abcam), SENP2 (1 : 1000; Abgent, San Diego, CA, USA), Flag (1 : 1000; Sigma), Myc (1 : 5000; Invitrogen, Carlsbad, CA, USA), Lamin B1 (1 : 1000; Bioworld, St Louis Park, MN, USA), HSP90B (1 : 1000; Bioworld), ubiquitin (1 : 1000, Abcam), acetylated lysine (1 : 1000; CST, Danvers, MA, USA) and GAPDH (1 : 10 000; Bioworld), followed by donkey anti-goat or goat anti-rabbit secondary antibody conjugated with HRP. Detection was performed using an enhanced chemiluminescence kit (Millipore, Billerica, USA).