![Figure 4. Molecular and functional characterization of SUMOylation-deficient WASp mutants. (A) EMSA with NF-κB, OCT1/2, and CTCF oligos performed on the nuclei isolated from TH1-skewed/TCR-activated WASp-deficient TH cells stably transfected with FL-WASp or PSM-WASp. Arrowheads indicate the location of the shifted bands. (B) Reciprocal, sequential immunoprecipitation (eg, WASp>SUMO1 sequence denotes 1stIP: WASp, 2ndIP: SUMO1) performed on the whole cell extract derived from primary TH1-skewed cells was resolved by western blotting the same gel with indicated antibodies. (C) The same gel shown in Figure 3G (Jurkat TH1-skewed cells, nuclear extract) was sequentially reprobed with the indicated antibodies. (D) MNase-ChIP. Chromatin fragmentation pattern and efficiency achieved with MNase treatment is shown in supplemental Figure 7. Chromatin enrichment profiles of the indicated proteins, at the 5′ untranslated region (−200 to −250 bp from first coding ATG, a region known to contain functional promoter elements) of the IFNG gene in TH1-skewed WASnull TH cells untransfected (UT) or stably transfected with Flag/Myc-tagged, full-length WASp (FL) or SUMOylation-deficient WASp mutants (PSM and V75M). For sequential ChIP, 2 rounds of conventional ChIPs were performed in the indicated sequence (FLAG>COMMD1, 1stChIP:FLAG, 2ndChIP:COMMD1; IgG>COMMD1, 1stChIP:IgG, 2ndChIP:COMMD1). The displayed ChIP values (mean ± standard error of the mean [SEM]) are percentage of total nuclear input chromatin and were derived after subtracting the background values obtained with isotype IgG antibody, the latter not shown for single ChIPs. Data were generated from 3 biologic replicates. NEM+/−, treated/nontreated with NEM.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/126/14/10.1182_blood-2015-05-646182/4/1670f4.jpeg?Expires=1735519775&Signature=DhtGirbugp7x3rj6YQx1jYEgEKA4vKYqZ56VwOV0XTq0uImK0GWtu~0D~LsSFZgzSpqrbvh-e~VxicvTK8b1bQcOI6d9mlAWOf98GBx4ABCBSF0fyrBeK3FEIUpTHv88f37LlUZBx-owpprPQPO43zGHUOnYO4FTSTjeqvh4XIrFYrXDk6jrrpRb0qxNiKGtBxLlaXiwurDq87zyOBEMd7bvTogevL6nhmKn6NSXtKItgSxrknFqvoG~prHiu68wHYolSMYOXJna-LapC~VOyPRS2IbhzfUqd4f-ASIn5BWtpkkypu2xe4SWdZzdcQwKZ7fq9edrt4VjxWLy8NP3bg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Molecular and functional characterization of SUMOylation-deficient WASp mutants. (A) EMSA with NF-κB, OCT1/2, and CTCF oligos performed on the nuclei isolated from TH1-skewed/TCR-activated WASp-deficient TH cells stably transfected with FL-WASp or PSM-WASp. Arrowheads indicate the location of the shifted bands. (B) Reciprocal, sequential immunoprecipitation (eg, WASp>SUMO1 sequence denotes 1stIP: WASp, 2ndIP: SUMO1) performed on the whole cell extract derived from primary TH1-skewed cells was resolved by western blotting the same gel with indicated antibodies. (C) The same gel shown in Figure 3G (Jurkat TH1-skewed cells, nuclear extract) was sequentially reprobed with the indicated antibodies. (D) MNase-ChIP. Chromatin fragmentation pattern and efficiency achieved with MNase treatment is shown in supplemental Figure 7. Chromatin enrichment profiles of the indicated proteins, at the 5′ untranslated region (−200 to −250 bp from first coding ATG, a region known to contain functional promoter elements) of the IFNG gene in TH1-skewed WASnull TH cells untransfected (UT) or stably transfected with Flag/Myc-tagged, full-length WASp (FL) or SUMOylation-deficient WASp mutants (PSM and V75M). For sequential ChIP, 2 rounds of conventional ChIPs were performed in the indicated sequence (FLAG>COMMD1, 1stChIP:FLAG, 2ndChIP:COMMD1; IgG>COMMD1, 1stChIP:IgG, 2ndChIP:COMMD1). The displayed ChIP values (mean ± standard error of the mean [SEM]) are percentage of total nuclear input chromatin and were derived after subtracting the background values obtained with isotype IgG antibody, the latter not shown for single ChIPs. Data were generated from 3 biologic replicates. NEM+/−, treated/nontreated with NEM.
