Characterizing physiologic WASp:SWI/SNF associations in vivo. (A) Mass spectrometry. Actual number of polypeptides of WASp-associated, chromatin-remodeling complexes (CRC) captured from cytosolic and nuclear fractions of TH1-skewed, primary TH (endogenous WASp) or Jurkat TH (Flag/Myc-tagged transfected WASp) analyzed by LC-MS/MS after immunoaffinity purification with anti-WASp, anti-FLAG/MYC (sequential 2-step purification), or -IgG antibodies, as described.3 Searches from 3 to 4 biological replicates were combined to generate a MultiConsensus report of peptides and proteins identified from the WASp proteome after applying the filtering criteria previously described3 (supplemental Figure 1). (B) Selective validation of MS-generated WASp-associated CRC proteome by co-IP. Protein complexes isolated by 2-step IP (1st:Flag, 2nd:Myc) from the nuclear fraction of TCR-activated, TH1-skewed, Jurkat TH stably expressing Flag/Myc-tagged WASp were resolved by sequential western blotting with the same gel with indicated antibodies. This image is part of the full gel image shown in Figure 2D. (C) MNase ChIP-qPCR. Chromatin enrichment profiles of the indicated proteins, at 5′UTR (promoter region) of the indicated genes in TH1-skewed, normal, or WASnull TH cells stably transfected with Flag/Myc-tagged, full-length (FL) WASp. For sequential ChIP, 2 rounds of conventional ChIPs were performed in the indicated sequence (eg, WASp>BAF47 denotes 1stChIP:WASp, 2ndChIP:BAF47; IgG>BAF47 denotes1stChIP:IgG, 2ndChIP:BAF47). The displayed ChIP values (mean ± SEM) are percentages 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 biological replicates. The genomic location of PCR primer/probes is shown in Figure 2C.
