Fig. 2.

The PCNA^S228I mutation differentially affects PCNA binding to PIP box-containing proteins. A) Schematic showing GST-PIP peptide constructs generated during this study. GST represented by rectangle, PIP box sequences shown in red, mutated residues shown in blue. Numbers relate to amino acid position in full length protein. B) GST-PIP pull down of His-S-PCNA^WT or PCNA^S228I. Figure shows Coomassie stained gel of representative pull down (top) and anti-PCNA western blot of the same samples diluted 1:20 (bottom). Amount of ‘input’ loaded for Coomassie is equivalent to 1%, ‘Glut. beads’ (Glutathione sepharose 4B beads) is equivalent to 25%. Molecular weight markers are indicated. C) Quantification of binding. Histogram shows amount of PCNA^S228I pulled down relative to PCNA^WT for indicated PIP constructs. Fen1 (n = 8), p21 (n = 8), p21* (n = 1), DNMT1 (n = 3), Cdt1 (n = 6). Error bars are standard deviation. D) GST-PIP pull down of His-S-PCNA^WT or PCNA^S228I for weak binders with 100 μg GST-PIP construct used per condition. Coomassie as B, quantification as C, n = 3.