Although currently having some trouble with myr-PKI in HT22 cells, myr-PKI is surely specific for PKA and much better than H89, which is, as Daniela Bertinetti pointed out above not at all specific for PKA but inhibits myriads of other kinases (see Davies SP et al., 2000, Biochem.J.).

As for your question, the so-called phospho-PKA-substrate antibody detects motifs that are phosphorylated by PKA, PKG and PKCs (CS correctly states this in their data sheet). CREB (at serine 133) is a substrate for PKA, but not a particularly good one. Sequences preferentially phosphorylated by PKA can be found in papers by Tegge, Dostmann & Frank or from JD Scotts lab. We have used the phosphorylation-dependent (pseudo-)shift in running of perilipin on the gel in adipocytes (Fricke et al., 2004 & 2005, Endocrinology) for Western-blot based PKA analysis.

A broader description of Western blot analysis of PKA phosphorlyation can be found in Wicht & Maronde et al., 1999, Histochem. Cytochem. or Maronde et al 1999, J. Neuroscience or Pfeffer et al., 1999 Mol. Pharm.).

Beside myr-PKI also Rp-cAMPS analogs are specific inhibitors for PKA, but may have effects on EPAC and cyclic nucleotide-dependent ion channels. At least they do not inhibit other protein kinases.

By Western Blot alone, I am not aware of a way that it can be done (at least not well). The P-PKA substrate antibody works well but it is by no means a specific enough readout of PKA activity that you could suggest that increased band strength is definitely due to PKA activity. There is a good affinity pulldown approach pioneered by Kathleen O'Connor, and we have used a combination of PKA IP followed by a homemade kinase assay using GST-CREB and phospho/total CREB Western blots. Other people have utilized specific phosphorylation sites on VASP (Ser157) to suggest increased PKA activity. While I am not sold that it is a good specific readout for PKA activity, it would probably be a good start if all you have access to currently is cell/tissue lysates.

If you have some specific PKA targets in mind, it might also be worth considering Phos-tag technology (developed here: Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T.

Mol Cell Proteomics. 2006 Apr;5(4):749-57 and developed further by the same lab in recent publications). Phos-tag is incorporated into SDS-PAGE gels and causes mobility shifts for phosphorylated isoforms, with distinct bands for each additional phophorylated residue carried by the protein. Gels can be blotted and probed with the antibody of interest. It might be possible to compare banding patterns for target proteins in control versus PKA inhibitor-treated cells. To date, we have used this to look at PKA sites using GST fusion proteins, but haven't tried blotting yet. Gels take some optimisation, but give strikingly clear results when it's up and running.

You could try phospho-specific antibodies to known PKA substrates but beware sites that can be targetted by other kinases. A good control is the effect of the H89 inhibitor (as well as a known agonist such as dibutyryl cAMP). A possible PKA substrates is CREB (Ser 133) for which there are good phospho-specific antibodies. Other known targets such as MLC and phospholamban are likely not expressed in adipocytes.

check for CREB phosphorylation (antibody available from CST)....

you can also use H89 treatment (PKA inhibitor) as a control

Involvement of different basophilic kinases on substrates are really cell and stimulus dependent. U can use phospho CREB / substrate motif antibody, but as Dipanjan suggested above, you will have to use inhibitor controls to make sure you are not measuring other basophilic kinases (PKC/PKD/MSK/MK2 etc)

Thank you very much for all the very helpful replies. I am taking on board all the suggestions. I think I will probably try the phosphoCREB antibody. Hopefully it will give me a reliable read-out, especially as I have conditions where I treat the cells with H-89 inhibitor. Kind regards.

I know, H98 is often called "PKA specific inhibitor", but that is not the case! The only PKA specific inhibitor is PKI (ProteinKinase Inhibitor, also called Walsh inhibitor). It is often used as the myr-PKI-14-22amide, which is cell permeable and really specific. For more informations see the reference:

Tracey E. Harris, Shanta J. Persaud, and Peter M. Jones (1997) "Pseudosubstrate Inhibition of Cycjavascript:lic AMP-Dependent Protein Kinase in Intact Pancreatic Islets: Effects on Cyclic AMP-Dependent and Glucose-Dependent Insulin Secretion" in BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 232, 648–651 (1997)

or the Research Gate Question "Is myr-PKI a good PKA inhibitor in cell culture? "

As having done live calcium imaging experiments, with both agents listed above. H89 is not a specific PKA inhibitor. It can knock down other phosphorylation pathways as well. PKI works extremely well and is fast about lower PKA activity. One must remember though some cells do not have high endogenous PKA phosphorylation so a functionality test to confirm would be advised.

Or if solely doing a western blot run a positive lane either your cells with a PKA stimulant to induce phosphorylation or a known isolate that you can use as a positive control

I found the paper about H89 targets, perhaps, this is helpful, too: Lochner, A., and J. A. Moolman. 2006. The many faces of H89: a review. Cardiovasc. Drug Rev. 24:261–274.

Although currently having some trouble with myr-PKI in HT22 cells, myr-PKI is surely specific for PKA and much better than H89, which is, as Daniela Bertinetti pointed out above not at all specific for PKA but inhibits myriads of other kinases (see Davies SP et al., 2000, Biochem.J.).

As for your question, the so-called phospho-PKA-substrate antibody detects motifs that are phosphorylated by PKA, PKG and PKCs (CS correctly states this in their data sheet). CREB (at serine 133) is a substrate for PKA, but not a particularly good one. Sequences preferentially phosphorylated by PKA can be found in papers by Tegge, Dostmann & Frank or from JD Scotts lab. We have used the phosphorylation-dependent (pseudo-)shift in running of perilipin on the gel in adipocytes (Fricke et al., 2004 & 2005, Endocrinology) for Western-blot based PKA analysis.

A broader description of Western blot analysis of PKA phosphorlyation can be found in Wicht & Maronde et al., 1999, Histochem. Cytochem. or Maronde et al 1999, J. Neuroscience or Pfeffer et al., 1999 Mol. Pharm.).

Beside myr-PKI also Rp-cAMPS analogs are specific inhibitors for PKA, but may have effects on EPAC and cyclic nucleotide-dependent ion channels. At least they do not inhibit other protein kinases.

Thank you Daniela and Erik for the very useful suggestions. Indeed I have gone off the idea of using phospho CREB to monitor the PKA activity for many reasons, but most importantly because I would be using cytosol for monitoring the activity and CREB is a localised in the nucleus. As I am working with adipocytes I think the most obvious PKA substrates are perilipin and HSL and my intention is to check the available phospho HSL antibodies. I know that a phospho perilipin antibody is not available therefore the band shift method is a good suggestion. For the inhibitors, H89 was a first step and I was going then to use something more specific.

I have got very good westerns with p-ser 133 CREB, p-ser-HSL, p-ser-perilipin (ABs from CST). But, I used an activator of PKA in my studies.

I work with the Cell Signaling ab #9621 and get good results for my protein when stimulating cells with forskolin. Preincubation with H89 also works for me - in HEK293 cells the PKA substrate ab usually doesn't recognize anything on my protein anymore. In addition, in HEK293 cells forskolin-dependent PKA activation leads to phosphorylation of VASP - the pAB also works nicely in these cells as a positive readout for stimulation.

Anybody knows if HSL is present in sperm?. I am looking to detect PKA activity by western blotting in these cells and I am doubting regarding the most idoneous substrate.

I have made an indirect detection of boar sperm PKA activity through western blot determination of its phosphorylated substrate DARPP32, which is present in spermatozoa. I perform a western for DARPP32 and, subsequently, I perform an stripping of the membrane, which is further probed with a total PKA antibody. The intensity ratio DARPP32/PKA would provide an indirect measurement of sperm PKA activity, at least in boar.

Hi!

can anybody please tell the time of treatment and medium for myr-PKI treatment for inhibition of PKA activity. I am using H9C2s and C2C12s and there after measuring calcium in the cells but I don't find any difference in calcium upon PKA inhibition. I am measuring the inhibition by Thermo PKA activity kit.