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2 RbAp46 and RbAp48 (for Retinoblastoma protein associated p48). Human p48 can bind to Histone H4 in the absence of CAF-1 p150 and p60. Results In this study, we have investigated the binding of Histone peptides to the Chromatin assembly factor I p48 subunit using MicroScale Thermophoresis. The peptides derived from the N-terminus of Histone H3 (N-terminal long [H3NL] and N- terminal short [H3NS]) as well as of the N- terminus of Histone H4 (N-terminal long [H4NL]). For the MST measurements the NT-647-labeled p48 and NT-647-labeled H3NL peptide were used at a constant concentration of ~ 50 nM. We first checked for the interaction of NT-647- labeled p48 with the histone H4 peptide H4NL. Fig. 2 NT-647-labeled p48 - H4 peptide H4NL capillary scans in standard and hydrophilic capillaries. Switching to hydrophilic capillaries prevented unspecific binding of the labeled protein to the capillary walls. The initial capillary scan in standard treated capillaries indicated unspecific binding of labeled p48 to the capillary walls at high concentrations of the H4NL peptide which could be prevented by choosing hydrophilic treated capillaries (Fig. 2). The resulting binding curve resulted in a calculated K d of 0.137 ± 0.062 µM (Fig.3). Fig. 3 Shape of the MST curves and binding curve of NT647- labeled p48 vs. H4NL peptide. The binding curve represents the data points from 1 measurement. The calculated K d is 0.137 ± 0.062 µM. We next checked the interaction of p48 with Histone H3 derived peptides. Fig. 4 shows the resulting binding curve for the H3NS peptide - NT-647-labeled p48 interaction with a calculated K d of 15.76 ± 2.18 µM. Fig. 4 Binding of H3NS peptide to NT-647-labeled p48 in standard treated capillaries: shape of the MST-curves and the resulting binding curve. The binding curve represents the data points from 3 measurements. The calculated K d is 15.76 ± 2.18 µM. We then measured the interaction of p48 with NT-647-labeled H3NL peptide which covers the complete N-terminus. The p48 protein stock used for this measurement was stored in a buffer containing 10% glycerol. To make sure, that no buffer effects are observed the dilution buffer for the H3NL-NT-647 labeled – p48 experiment was prepared accordingly making sure that the final glycerol concentration was 5 % in all samples. Fig. 5 shows the resulting binding curve and the shape of the MST curves. The calculated K d was 4.2 ± 0.93 µM, which indicated a slightly higher affinity of the H3NL peptide for p48 compared to the H3NS peptide. This K d was in good agreement with the K d determined for the H3NL – p48 interaction when p48 was labeled, which gave a dissociation constant of 6.95 µM (data not shown).

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