need to inhibit integrins but nor sure about the RGD peptides are potential enough. Which commercial RGD peptide would be better?
i'll observe if inhibition of integrin signaling can reduce ROS generation. And need to prove how it is happening.
Need to apply soluble RGD. Please share your knowledge regarding this.
Best
It depends on the integrin of interest. If your integrin of interest is an RGD-binding integrin then more than RGD-inhibition wil give you the desired inhibitory effect. Been shown many times blocking integrin signaling with an RGD peptidomimetic compound is quite effective in blocking integrin signaling, for example in blocking TGF-beta activation or cancer cell invasivness (alternatively you could always employ other inhibitors along with your RGD-inhibition to show that ROS generation (for your experiment) is reliant only on the RGD signaling and not some other pathway you've deemed important ie there's no added benefit from employing other inhibitors as well). Only other thing to keep in mind is integrin activation and signaling can be activated quickly and once the pathway gets set into motion it can propagate quickly, so you may need to adjust your incubation times accordingly or always keep the RGD peptidomimetic in your media during experiments
Actually I am going to use Collagen-I, to which the receptor is Integrin alpah2beta1. I am in need of the information that if I use RGD from any commercial source then how strongly it could inhibit the Integrin signaling. I have found that cyclic RGD is good enough comparing the action to linear form. I am in a lack of info condition.
yes, if you're just trying trying to block the RGD-binding domain of an integrin, any RGD peptide really should be fine, it's just a three amino acid peptide so very simple. then to be absolutely sure that this is happening due to the RGD binding domain on Integrin-a2b1 you could use a specific monoclonal to that integrin dimer pair and determine if there's any difference between antibody blocking and the RGD peptidomimetic. but yes, in my opinion, as far as the RGD peptides go, purchasing one from a respected vendor and following protocol will certainly provide the inhibition you're looking for!
Thank you again Anthony. I would like see the NOX profile after confirming the inhibition. What would be the best way to do that? I am not sure if I could avail microarry, so looking for any alternative way to get the specific result.
I guess there could be a few different avenues you could take here. You could look at ROS levels with DCFH-DA (easily purchased from invitrogen) which you can incubate with your cells and if there are oxidants present it will fluoresce blue and you can just read it on a spectrophotometer and quantified. Then I would think to ensure the differences in ROS are due to Integrin inhibition upstream of NOX you could repeat the experiments +/- different NOX siRNA, shRNA or inhibitors. Many people also use the lucigenin based assays as well which seem to be quite easy. Good luck!
some links: http://rusynlab.unc.edu/publications/course_data/Wang%20et%20al%202011.pdf
http://www.jneurosci.org/content/30/11/3933.full.pdf
I am much obliged dear Anthony. In my lab we actually use DCFH-DA, Luceigenin and MitoSox assay. No problem with that I guess.
That was very helpful indeed.
Regards
Just to clarify, if your integrin ligand binding to collagen is inducing ROS, then blocking with RGD should not inhibit.
Integrin-collagen interactions are not canonically mediated via a RGD sequence.
http://jcs.biologists.org/content/119/19/3901.short
Also to add that monoclonal antibodies are great tools for these sort of assays.
http://jcs.biologists.org/content/122/22/4009.short
Best wishes.
Dear Jonathan,
Thank you very much. I was dealing with DDR2 (a collagen receptor) and coating the wells with collagen-I was showing increased ROS generation in DDR2 negative cells. Now I would like to see whether DDRs or Integrins are responsible for the ROS generation. Need to inhibit integrins, and I am not sure if RGDs are the best choice.
Antibodies would be great choice indeed.
Best Regards
I would suggest that RGD should not be your first choice for this question. It may be a great control though - if you found a collagen peptide that worked. I am not sure where you would purchase integrin binding collagen peptides but I am sure the literature would help - I would look to see what Richard Farndale had done http://www.bioc.cam.ac.uk/people/uto/farndale
Regards
Info regarding RGD confused me a bit. Opposite info are coming up. RGD can inhibit and it can't. Definitely I'll look for something that has direct evidence. And thank you so much for the link. I am sure I could get the best out of it.
Kind regards
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