Click it cell reaction cocktail

MDA-MB-231 cells growing on the cover slips were washed once with PBS and then the cells were covered with PBS. The cells were treated with DMSO or LBL1 for 20 min followed by LBL1-P for 20 min. After UV-irradiated for 5 min, the cells were fixed with 3.7% formaldehyde and permeabilized with 0.5% triton X-100. After the cells were washed with 3% BSA in PBS, the cells were incubated with Click-iT^® Cell reaction cocktail (Life Technologies) supplemented with rhodamine-N₃ for 30 min at room temperature. Then the cells were washed with 3% BSA in PBS before being incubated with anti-LA (mouse, Sigma, cat# SAB4200236, 1:1000) overnight. The cells were then incubated with Alexa Fluor 488 donkey anti-mouse secondary antibody for one hour at room temperature and the coverslips were mounted as above. The coverslips were then mounted in ProLong Gold AntiFade Reagent with DAPI (Life Technologies) and the images were acquired with a fluorescence microscope ApoTome (Zeiss). For colocalization analysis, the Z-stack of images were reconstructed and colocalization Pearson correlation coefficients were determined using the Coloc module in the Imaris software package (Bitplane).