The APIM sequence in XPA is sufficient and necessary for interaction with PCNA.
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Abstract
<p>(A) Sequence alignment of the APIM sequence in XPA (aa 161–170 in human XPA) from different species compared with the APIM sequence in hABH2. The colors are given by Clustal X. (B) Dot blot with the human XPA APIM-peptide. The hABH2 APIM-peptide and its mutant are included as positive and negative controls, respectively (also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049199#pone.0049199-Gilljam1" target="_blank">[27]</a>). Grey lines: dots from the same blot. (C) Images of YFP-tagged XPA<sub>161−167</sub> co-expressed with CFP-tagged PCNA in live cycling HeLa cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (D and E) N<sub>FRET</sub> measurements in HeLa cells. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET) (D) and 700 (YFP), 800 (CFP), 700 (FRET) (E). CFP/YFP (vectors only) and CFP-PCNA/YFP-PCNA were used as negative and positive controls, respectively (mean ± SEM, n = 24–53 in D and n = 10–34 in E). (F) Overexpressed tagged proteins in live cycling XPA<sup>−/−</sup> cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (G). N<sub>FRET</sub> measurements in XPA<sup>−/−</sup> cells. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET) (mean ± SEM, n = 25–66). The P-values (D, E and G) are derived by unpaired t-test.</p