Equal numbers of subconfluent MCF7 cells expressing GFP alone, cavin1-GFP, cavin2-GFP, cavin3-GFP, and CAV1-GFP were seeded on coverslips. Twenty-four hours later, cells were subject to UV-C exposure for 2 min without media. Complete medium lacking phenol red was added to the cells that were left at 37°C to recover. LDH release assay was measured in triplicate samples from 50 μl of conditioned media expressing cells using the Cytotoxicity Detection KitPLUS (LDH) from Sigma-Aldrich according to the manufacturer's instructions. Post-nuclear supernatant from UV exposure cells was also prepared and subjected to western blot analysis with antibodies to BRCA1 (WB 1:500), GFP (WB 1:3000), and Tubulin (WB 1:5000). For knockdown experiments of cavin3 and BRCA1, after 72 hr of knockdown, cells were left untreated or were further transfected with BRCA1-GFP or cavin3-GFP overnight, respectively, and were then subjected to UV exposure 2 min and a recovery time of 6 hr. LDH release was then measured from the cell supernatant in triplicate as indicated in the respective figure legends.

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.