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. 2021 Feb 2;12:751. doi: 10.1038/s41467-021-21025-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — The time courses of paCaMKII (a) and light-insensitive super-dark paCaMKII (SD) mutant (b) dephosphorylation at T286 after light illumination. Dissociated hippocampal neurons were infected with AAV-DJ encoding Flag-His×6-paCaMKII or mutant under CaMKII promotor were illuminated with blue light for 3 min. Right after illumination (at 0 min in the figure), samples were incubated in the dark for the indicated time. c Quantification of a and b. The number of samples (n, biologically independent) at each point is as the following. For paCaMKII, before, n = 8; 0 min, n = 8; 1 min n = 4; 3 min, n = 4; 10 min, n = 4; 30 min, n = 8. For paCaMKII(SD), before, n = 6; 0 min, n = 6; 1 min n = 4; 3 min, n = 3; 10 min, n = 4; 30 min, n = 6. The band intensity of before light was used for normalization. The band intensities of pT286 were divided by those of total. The data are presented as mean ± SEM. d Selectivity of paCaMKII. Dissociated hippocampal neurons were infected with AAV-DJ encoding Flag-His×6-paCaMKII under CaMKII promotor were stimulated with no light (lane 1), blue light for 5 min (lane 2), or 20 µM NMDA for 2 min (lane 3). The left panel shows the autophosphorylation of paCaMKII and CaMKIIα/β, and the right panel shows the expression of the respective proteins. Light illumination selectively activates paCaMKII, but not CaMKIIα/β (lane 2 in the left panel), whereas NMDA stimulation activates paCaMKII and CaMKIIα/β (lane 3 in the left panel). e Incorporation of paCaMKII into CaMKIIα/β oligomer. Dissociated cortical neurons with no transfection (lanes 1 and 2) and AAV-DJ encoding Flag-His×6-paCaMKII transfection (lanes 3 and 4) were lysed and pulled down with Ni sepharose followed by immunoblotting with CaMKIIα/β antibody. In lane 3, Flag-His×6-paCaMKII was pulled down, and subsequent western blotting identified that CaMKIIα/β are incorporated, suggesting that paCaMKII, CaMKIIα, and CaMKIIβ form oligomer. Source data are provided as a Source Data file.