sumo hybridomas sumo1 21c7  (Developmental Studies Hybridoma Bank)

Journal: Genes & Development

Article Title: ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation

doi: 10.1101/gad.345843.120

Figure Lengend Snippet: SUMOylation of SATB2 during ESC differentiation toward ectodermal progenitors. ( A ) Immunoblots detecting SATB2, NANOG, and TUBULIN in wild-type ( Satb2 wt ) and Satb2-deficient ( Satb2 Δ/Δ ) ESCs, cultured in LIF or with retinoic acid (RA) for 2 or 4 d. Blots were generated with ESCs from Satb2 wt clone 8 and Satb2 Δ/Δ clone 2. Data are representative for three independently derived Satb2 wt clones (8, 24.1, and 24.3) and Satb2 Δ/Δ clones (2, 32, and 64). Satb2 Δ/Δ ESCs were generated by treatment of Satb2 fl/fl Cre ERT/+ ESCs with 10 μM tamoxifen for 5 d. All clones were cultured on fibronectin-coated dishes before replating onto gelatin-coated dishes for differentiation. ( B ) qRT-PCR analysis to detect Satb2 and Nanog transcripts in LIF-cultured or RA-treated (d2 and d4) Satb2 wt and Satb2 Δ/Δ ESCs. Values are derived from three clones each and are calculated relative to those of Satb2 wt clone 24.1 in LIF and normalized to Pgk . ( C ) Alkaline phosphatase staining and quantification of Satb2 w t and Satb2 Δ/Δ ESCs cultured in LIF or RA for 3 d. The percentages of undifferentiated, differentiated, and mixed-type colonies are indicated in the bar plot. Images are representative of two independent experiments carried out with each clone. Values in B and C are expressed as the combined mean ± SD of all clones from either genotype. t -tests were carried out relative to Satb2 +/+ cells. Significance levels are as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( D ) Immunoblots detecting SATB2 and RanGAP in ESCs, cultured in LIF or with retinoic acid (RA) for 2, 4, or 6 d. Whole-cell lysates were purified in the presence (+) or absence (−) of the SUMO-isopeptidase inhibitor NEM (10 mM). SUMO-modified forms of SATB2 and RanGAP are marked. RanGAP was used as a positive control for SUMOylation, and Tubulin was used as a loading control. ( E ) Immunoblots of endogenous SATB2 that was immunoprecipitated (IP) with mouse anti-IgG, bead-coupled anti-SUMO1, or anti-SUMO2 antibodies and eluted with SUMO1 or SUMO2 peptides. Cell culture conditions were as in A . The asterisk indicates the IgG bands of antibodies used in the IP. ( F ) Immunoblot analysis of whole-cell extracts from wild-type ESCs randomly differentiated (−LIF) and toward ectoderm using two distinct protocols. Blots were probed with antibodies to detect indicated proteins. Ectoderm differentiation was induced by LIF withdrawal and addition of RA, or by the addition of FGF2, FGF8, and SHH. ( G ) Schematic representation of SATB2 protein, highlighting the SUMO consensus motifs at K233 and K350 in SATB2 wt and the arginine substitutions (K → R) in the double-mutant SATB2 K → R . ( H ) Immunoblots of an in vitro SUMOylation assay using recombinant purified SATB2 wt , SATB2 K → R , and increasing amounts of recombinant ZFP451protein (amino acids 2–246), comprising SUMO E3 activity . ( I ) Coimmunoprecipitation of SATB2 to detect interaction with ZFP451 and PIAS1 in Satb2 wt (wt) or Satb2 K → R ESCs cultured in LIF or in RA for 4 d. Immunoblots are representative of three or four independent experiments using wt clones DV3 and C2 ( A,B ) and Zfp451 +/+ clone 14 ( B ). In vitro SUMOylation immunoblot is representative of three independent experiments.

Article Snippet: SUMO hybridomas SUMO1 21C7 and SUMO2 8A2 were developed by M. Matunis and obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development of the National Institutes of Health and maintained at Department of Biology, The University of Iowa ( ; http://dshb.biology.uiowa.edu ).

Techniques: Western Blot, Cell Culture, Generated, Derivative Assay, Clone Assay, Quantitative RT-PCR, Staining, Purification, Modification, Positive Control, Immunoprecipitation, Mutagenesis, In Vitro, Recombinant, Activity Assay