XPA colocalizes and directly interacts with PCNA in replication foci

<p>. (A) Overexpressed tagged proteins in live cycling HeLa cells. (B) Immunostained HeLa cells. The intensity of α-XPA and α-PCNA along the line in the merged picture is illustrated in the graph. The inserts show an enlargement of the area close to foci 3 and 4. (A and B) Bar: 5 µm. (C) iPOND from cells labeled with EdU (pulse) before fixation. One sample was additionally followed by a chase in thymidine-containing medium (pulse-chase). The WB shows proteins captured due to EdU proximity. The upper and lower panels are from individual iPOND experiments. All bands within one panel (black frame) are from the same WB, lanes and rows are separated by grey lines (also in D and E). (D) Co-IP of endogenous XPA from HeLa cells stably expressing YFP-PCNA using α-YFP beads. SF: soluble fraction, CF: chromatin-enriched fraction, Y: YFP (negative control), Y-P: YFP-PCNA. (E) Co-IP of endogenous XPA from untransfected HeLa cells using α-PCNA beads (pulling down endogenous PCNA). IP with α-YFP was used as control for unspecific binding to the beads. (F) Normalized FRET (N_FRET) measurements in HeLa cells. CFP/YFP (vectors only) and CFP-PCNA/YFP-PCNA were used as negative and positive controls, respectively. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET). The P-value is derived by unpaired t-test. Data presented is from three independent experiments (mean ± SEM, n = 55–75).