Journal: Proteomes

Article Title: Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

doi: 10.3390/proteomes6040042

Figure Lengend Snippet: In vitro phosphorylation of α4/β2 nAChRs by CaMKII or PKA. The α4 and β2 nAChR subunits were co-expressed in HEK cells, isolated by immunoprecipitation, and subjected to mass spectrometry. Phosphorylation level was normalized to total subunit protein. ( a ) At baseline, there was a high level of phosphorylation of S470, S530, and S540 on the α4 subunit, and incubation with lambda phosphatase dephosphorylated S540 and S543 to undetectable levels. ( b ) Incubation with CaMKIIα in the presence of calcium and calmodulin increased phosphorylation of T417 and S468 on the α4 subunit significantly. ( c ) Incubation with PKA in the presence of cyclic AMP increased phosphorylation of S470, S491, and S521 significantly. * p < 0.05; *** p < 0.005. Error bars represent standard error of the mean; n = 6/condition.

Article Snippet: The remaining three groups were harvested in the absence of phosphatase inhibitors, and immunoprecipitated receptors were subject to in vitro dephosphorylation with purified lambda phosphatase (New England Biolabs, Ipswich, MA, USA; all but baseline group received this treatment).

Techniques: In Vitro, Isolation, Immunoprecipitation, Mass Spectrometry, Incubation