Fig 2: Data mining of FLAG-SMARCAD1 affinity purification from human cells reveals co-purification of architectural proteins. (a) Flow chart of the experimental approach used in [57]. IgG and FLAG pulldowns were performed, and peptides were quantified using label-free mass spectrometry. Identified peptides for the proteins we validated here are shown in Supplementary Figure S1. (b) The six polypeptides of the hTFIIIC complex and their known interactions according to the STRING physical network. TFIIIC complex division into two sub-complexes is indicated; modules A and B bind A and B box DNA loci, respectively [60]. Shown in bold are the five subunits that were identified by mass spectrometry as associated with tagged, overexpressed SMARCAD1 by [57].

Fig 3: Gene expression analysis after depletion of Smarcad1 in mESCs. (a) Experimental outline for testing the ability of SMARCAD1 to affect the transcription of TFIIIC- and TFIIIC-regulated loci. Two ESC lines were investigated, namely PGK12.1 and E14. SU, subunits. (b,c) RT-qPCR-based characterization of the relative expression of (b) TFIIIC subunits and (c) selected loci corresponding to different TFIIIC binding sites in control or Smarcad1 knockdown ESCs. Gene expression is normalized to the average of two housekeeping genes and is presented as mean ± S.E. (error bar) of three technical triplicates. Biological replicates of datasets for b and c are shown in Supplementary Figure S2.

Fig 4: Validation of architectural proteins as bona fide SMARCAD1-associated proteins by reciprocal co-immunoprecipitation of endogenous proteins. (a) Flow chart of the experimental approach. (b) Endogenous co-immunoprecipitation experiments (n = 2–4) from nuclear extracts from HeLa cells with anti-SMARCAD1 or anti-TFIIIC antibodies (subunits 63 and 90) analyzed by Western blot with the indicated antibodies. Inputs, 9% of the nuclear extract, are from the same blot as the IP. IgG, immunoglobin G. KAP1, a known binding partner of SMARCAD1, was analyzed in parallel (positive control).

Fig 5: Total (T), soluble (S for supernatant), and chromatin (P for pellet) fractions were prepared from control (Ctrl) and stable Smarcad1 knockdown (KD) ESCs (PGK12.1) and analyzed by Western blot with indicated antibodies. GAPDH and histone H3 were used as control for cytoplasm- and chromatin-bound fractions, respectively. Molecular weight markers are indicated in kDa. For TFIIIC90, bands of different mobility were detected, likely corresponding to differently modified versions of the protein.