Detection reagents orange

To visualize the subcellular localization of the ILK–RhoT1 interaction, MDA-MB-231 cells were stably transfected with pPB vectors encoding mEmerald-ILK and V5-RhoT1. To avoid mislocalization of ILK by its excessive expression, mEmerald-ILK cDNA was placed downstream of the TRE3G promoter, and expression was induced by the addition of 10 nM doxycycline 1 h after plating. After 48 h, cells were sequentially fixed with 2% PFA and −20 °C methanol, and blocked with MAXblock as described above. After washing in PBS, cells were incubated with an anti-ILK mouse mAb (1:50) and anti-V5 rabbit mAb (1:50) in 5% BSA/PBS for 16 h at 4 °C. PLA reactions were performed using Duolink® In Situ PLA Probe Anti-Mouse PLUS, Anti-Rabbit MINUS, and Detection Reagents Orange (Sigma), following the manufacturer’s instructions. After the ligation and amplification reactions, cells were further incubated with the anti-TOMM20 antibody conjugated with Alexa 405 (1:100) and DRAQ5 (1:5000) in Duolink Antibody Diluent (supplied in the kit), to visualize mitochondria and nuclei, respectively. Specificity of the antibodies used for PLA was confirmed by immunostaining performed at the same concentrations.