FAC protein is decreased in nuclear fractions of FA cells compared with normal and corrected FA cells. Cells were fractionated according to the method of Tsuda et al,27 and the nuclear lysates (50 μg each) were analyzed by SDS-PAGE. FAC protein (arrow) was detected by probing with FAC1 antisera (A and C) or stripping and reprobing with FAN2 antisera (B and D). A and B and C and D represent independent experiments where cells in columns 3 and 4 were transduced by pseudo-typed viruses encoding neo or FAC, respectively. Column 1, JY; column 2, HSC536N; column 3, HSC536N/neo; column 4, HSC536N/FAC. The blots were stripped and reprobed with β tubulin as shown in the bottom 2 panels, and the signals for nuclear and cytoplasmic β tubulin were estimated by densitometry. These fractionation quality controls are corrected for cell number, and the numbers shown are the percent of cytoplasmic protein contained in the nuclear fraction. Thereby, we estimate that the artifactual component of the signal detected for FAC in the nuclear fraction of JY cells (for A and B) is only 6%.