MCF10A, HCC1937, HCC1937 BRCA1 and CAL51 cells were cultured alone or transfected with pCDNA3, BRCA1a, BRCA1a Mut#1, BRCA1a Mut#4, BRCA1a Mut#9 or EGFP-Antisense Ubc9 for 24 hours in six-well plates onto glass coverslips overnight. The cells were washed and fixed in icy methanol for 5 minute, and blocked using 10% BSA for 60 min, followed by primary polyclonal Rabbit anti-caveolin-1 antibody 1:150, primary polyclonal anti-SIRT1 antibody (Santa Cruz),l:150 diluted in 1.5% BSA made in PBS at 25°C for 1hr and Alexa 488 goat anti-Rabbit/Alexa 568 goat anti-mouse (Molecular Probes) diluted in 1.5% BSA/PBS for 50 min and stained (Hoechst 33258, Pentahydrate, Life technologies). The cover slips were mounted with Vectashield mounting medium for fluorescence (H-1000 from Vector). The stained cells were examined by LSM 700 Confocal Microscope, equipped with 63× oil Ph immersion objectives. Composite filter cubes were used for the 488–405 as described previously [27 (link)].