Figure 1.

Immunofluorescence analysis of PCNA complex triggered by γ-ray irradiation in NHDF cells. Cells synchronized at G₁ stage were irradiated (5 Gy), untreated or treated with buffer I and fixed after 30 min in a mixture of acetone:methanol (1:1). The cells were indirectly immunostained with a primary mouse monoclonal antibody to PCNA and a FITC-conjugated secondary antibody to mouse IgG2a. In order to determine whether or not the total cellular PCNA level is elevated by IR, the cells were immediately fixed in acetone:methanol without the extraction of soluble proteins (A). Immunofluorescence of PCNA in control and γ-treated cells without buffer I extraction (a) and the western blot detection of PCNA in the total cellular proteins isolated by RIPA buffer (b) show similar levels of PCNA. A histogram shows the intensity of PCNA band in control and irradiated NHDF cells (c). NDHF cells were extracted with buffer to reveal the chromatin-bound PCNA formation (B). The cells stained with DAPI are given in gray scale.