Hello,
I'm trying to see whether, in renal cancer cells in vitro, bosutininb is able to inhibit Src activation by looking at phosphoTyr416 Scr by western blot. I've tried 1uM bosutinib at different incubation times (from a few hours to 48h), but I hardly see any reduction at all. The antibody I'm using is Cell Signaling #2101.
I was wondering what experimental protocol approach would give me better results, or whetherI could test other Src targets to verify bosutinib activity. Could it be that bosutinib is inhibiting other Src-family targets in my model (as the antibody recognises all Src-family phospho-proteins)?
Appreciate any suggestions and help, thanks!
Hello, we have been using cell signaling 6943. This antibody is very sensitive. 1uM is very high conc. already. I would suggest using a potent SRC inhibitor such as dasatinib (100nM-1uM) in a 24Hr experiment to use as positive control.
Hi Jordi,
CST 2101 (Rabbit monoclonal anti-pY416 SFK) is a very good antibody and we have been consistently using it since CST introduced this rabbit monoclonal in the market. The problem with detecting a decent inhibition of pY416 SFK lies on the degree of SFK phosphorylation itself. Does your renal cancer cells show a decent pY416 SFK phosphorylation compared to the normal control? Before expelling bosutinib, try challenging control cells to hyposmotic solution (200mOsm) for 1, 2 and 5 min and see the extent of pY416 SFK phosphorylation and then try to block it with bosutinib at your working concentration (1uM). Preincubate the cells for at least 20 min before exposing the cells to hyposmotic solution containing bosutinib. Ideally pY416 SFK inhibition is best achieved with the inhibitor if the degree of phosphorylation is at least >2 fold. When the SFK phosphorylation is little then it becomes difficult to block by using any of the SFK inhibitors.
Another problem with SFK inhibitors is that they sometimes show SFK phosphorylation at Y416 when used alone. The cells do not usually tolerate those inhibitors very well and too long incubation may cause significant cell death associated with increased pY416 SFK phosphorylation. If my advice fails, then you can certainly use dasatinib, PP2 or SU6656.
Instead of targeting the active site pY416 phosphorylation you may think considering the inhibitory Y527 on SFK (Phospho-Src (Tyr527) Antibody, CST #2105). If your diseased cells show a decrease of pY527 band signal then ideally bosutinb will be able to restore the phosphorylation level back to control values.
Hope that helps!!
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