Detection of an interaction between BRCA1 and the cavin or CAV1 proteins was assessed using the Duolink II Detection Kit (Sigma-Aldrich) according to the manufacturer's specifications. The Duolink In situ PLA Probe Anti-Rabbit MINUS (Sigma-Aldrich, DUO92005, RRID:AB_2810942) and Duolink In situ PLA Probe anti-Mouse PLUS (Sigma-Aldrich, DUO92001, RRID:AB_281039) and Duolink In situ detection reagents Orange (DUO 92007) were used in all PLA experiments. The primary antibodies used were mouse monoclonal GFP (1:500) and rabbit polyclonal BRCA1 (1:200), rabbit cavin3 (1:200) and mouse PCNA (1:100), rabbit cavin3 (1:200) and mouse Aurora Kinase (1:100), rabbit cavin3 (1:200) and Flotillin (1:100), and cavin3 (1:200) and mouse cavin 1 (1:100). The signal was visualized as a distinct fluorescent spot and was captured on an Olympus BX-51 upright Fluorescence Microscope. The number of PLA signals in a cell was quantified in ImageJ using a Maximum Entropy Threshold and Particle Analysis where 50 cells in each treatment group were analyzed from at least three independent experiments.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.