Chiara:  If you have a soluble form of your receptor, this will be the best "inhibitor" to prevent the cytokine from being scavenged.  Other generic agent for endocytosis will be mild base such as NH4OH which raises the pH of endosomes thus preventing it from being functioned. Hope this helps.

Hi Chiara,

You can also try specific blocking antibodies to inhibit receptor function or receptor-cytokine interaction. If receptor  itself or its interaction with the cytokine are blocked and scavenging continues it may be using an alternative mechanism.

Good luck!

Hi there,

The problem with dynasore is that it blocks both clathrin-dependent and clathrin-independent endocytic pathways involving dynamin.  

The best way to assess whether your ligand is scavenged by endocytosis of the ligand-receptor complex is to knockdown the receptor and clathrin, either alone or in combination, and then measure both surface-bout and free ligand levels.

Good luck with your experiments,

Metello

You might consider Pitstop2 (abcam). I have no personal experience with it but Volker Haucke's group showed that it is specific and  blocks clathrin mediated endocytosis  at all stages by binding to the terminal domain of clathrin heavy chain. The paper is by Lisa von Kleist et al. and her thesis on it is also available on-line.

Chiara, one method to inhibit CME is not sufficient, you will need different ways, which could include dynasore or pitstop, but you probably also need dominant negative dynamin expression or clathrin shRNA.  However, none of these specifically addresses whether your cytokine comes in through RMA.  Fluids also come in through CCV.  Do you know what the receptor is? can you knock it down with shRNA?  RME should display saturation kinetics, whereas fluid uptake will not.  Good luck

Thank you all. Actually I tried Dynasore by Sigma. I did not see any inhibition of endocytosis, verified by ELISA quantification of the ligand into the supernatant (which decreases with the time, because of the scavenging by the receptor).

Anyway, I did not find a precise protocol for using Dynasore with HeLa cells, and somehow I feel to not have worked with the right conditions. Does anyone know which are the best conditions for inhibition?? I mean something like overnight pretreatment with Dynasore, starvation, confluency conditions...

thank you!

There can be better reasons why you saw ligand reduction in the presence of Dynasore. Dynasore action can be reduced by serum. So instead of 85uM I use 100uM which worked best. Second, Dynasore does not prevent pinocytosis. So the cells may still take up ligands in a non-selective way by gulping liquid.

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