How can I inhibit clathrin mediated endocytosis? - Answered

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I'm currently studying EGFR trafficking in various cell lines. EGFR is quickly internalized after EGF stimulation and I read somewhere that this process is, in most cell types, clathrin and dynamin mediated. I would like to check if in my cell lines this process is also clathrin and/or dynamin mediated. What is the easiest way to inhibit clathrin and/or dynamin mediated endocytosis? Are there small molecule inhibitors? siRNA?

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  Vibor Laketa Wednesday, 10 July 2013 - 19:08 UTC

  Two options come to my mind:

  1.use siRNAs against clathrin heavy chain or dynamin2 (works well in my hands to inhibit EGFR endocytosis)
  the drawback is, of course, that you have to wait 2-3 days for siRNAs to induce maximal downregulation and you will never know which cells are transfected with siRNA and which not. You will probably not see 100% block but you will see inhibition of endocytosis.

  2. small membrane-permeable molecule that inhibit dynamin or clathrin....
  for dynamin you could use dynasore (not sure you can buy it, you can definitely ask the people who published it http://www.lifescience.net/publications/239/dynasore-a-cell-permeable-inhibitor-of-dynamin/) we used it and sometimes it worked and sometimes did not, it seems if the cells are too confluent than it does not work

  for clathrin you can use pitstop1 or pitstop2 (http://www.lifescience.net/publications/240/role-of-the-clathrin-terminal-domain-in-regulating/)
  I have no experience with these compounds but it seems they work nicely