BRCA2-GFP is oligomeric in cells. (A) Fluorescence intensity bleaching traces (blue line) from six fixed cells were acquired by integrating the fluorescence intensity of 5 × 5-pixel windows centered around an intensity maximum and corrected for background using the mean value of the surrounding 9 × 9 frame (gray; see inset). (B) A histogram of the steps (n = 360) obtained from the step-function fit (red line in A) was used to estimate the mean step size by fitting the distribution with a Gaussian function. This mean step size, as mean intensity per GFP, was used to determine the number of BRCA2-GFP molecules per fluorescent object in two different ways. (C) Steps larger or equal to this mean step size were counted; steps more than twice the size of this mean were calculated as two BRCA2-GFP molecules (n = 90 traces with at least one BRCA2-GFP). (D) Alternatively, the difference between initial and final intensity in the bleaching traces (n = 118 with at least one BRCA2-GFP) was divided by the mean step size to determine the number of molecules per BRCA2 cluster. The data shown are representative of multiple experimental repeats (n = 3).