hnRNP K is associated with SETDB1 and KAP1 in mESCs.

(A) IP scheme to identify SUMO-dependent binding partners of SETDB1 and silver stained gel showing protein content of the indicated fractions. The nuclear extract input (NE), negative control IP (IgG), SETDB1 IP (α-SETDB1), and the flow-through (FT), 0.25 M KCl and 0.5 M KCl fractions from the anionic column are shown. (B) Western blot of SETDB1 and SENP1 in the indicated fractions and IP. (C) Silver stained gel of FLAG-SETDB1 IP and western blot of FLAG-SETDB1, KAP1 and hnRNP K in immunopurified FLAG-SETDB1 complexes isolated from tamoxifen-induced Setdb1 KO mESCs stably expressing 3XFLAG-Setdb1 (KO+FLAG-Setdb1) or negative control uninduced Setdb1 conditional KO cells (cKO). Complexes were immunoprecipitated with FLAG antibodies and specifically eluted with 3XFLAG peptide. Asterisk marks a non-specific band. Cropped band at bottom of hnRNP K blot is IgG heavy chain (~55 kDa). (D) Co-IP assay of endogenous KAP1 and hnRNP K with SETDB1 from mESC nuclear extracts in the absence or presence of 10 mM SENP inhibitor (NEM). Note that hnRNP K is detected in mESCs as two bands at ~65 kDa and ~60 kDa, the larger of which represents full-length hnRNP K and the smaller is a splicing isoform hnRNP J, also produced from the Hnrnpk gene [77]. The hnRNP K isoform is associated with SETDB1. ‘NE’ represents ~10% of nuclear extract input and ‘IgG’ is the negative control IP. (E) Co-IP assay of endogenous SETDB1 with KAP1 or hnRNP K from mESC nuclear extracts in the presence or absence of NEM, as in (D).