Hello all,
I would like to investigate whether my molecule activates lipolysis via PKC. The PKC activates the MAPK pathway which phosphorylates the hormone sensitive lipase (HSL) at Ser600. However, I can only ever see in papers antibodies against for example Ser660 or Ser565. However, these are only phosphorylated at the PKA or AMPK pathway.
Has anyone worked on this topic before and can help me?
Thanks a lot
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Lisa,
If you can't get the specific antibody for your protein, you may try to tag the protein with a tag (e.g. flag, GFP, and HIS tag). Then you can do western blot with an anti-tag antibody.
If you could make an HSL mutant with S600A (something like this), then you could compare the size by western blot with an anti-HSL antibody. the S600 phosphorylated HSL should run slower than the S600A control.
Check https://www.cellsignal.com/products/primary-antibodies/phospho-hsl-ser660-antibody/4126
Experiments in murine adipocytes have demonstrated that Ser563, Ser659, and Ser660 are the major protein kinase (PK)A phosphorylation sites, which are essential for the translocation of HSL to the lipid droplet surface and for stimulation of HSL ( 9 ). I got the information from the paper below:
https://www.jlr.org/article/S0022-2275(20)36702-X/pdf
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