The Journal of Neuroscience, December 27, 2006, ():
Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation
J. Neurosci. Misonou et al.
26: 13505
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Quantitative immunoblot analysis with the phosphospecific antibodies
(A) Linearity of immunoblotting with S563P and S603P. Different amounts of RBM (5-40 μg as indicated) were separated by SDS-PAGE and probed with the phosphospecific antibodies. Numbers to left indicate the mobility of molecular weight standards in kDa.
(B) Densitometric analysis of linearity of immunoblotting. Optical densities of the immunoreactive bands were measured and plotted against the amount of the total protein loaded. Parallel analyses of 20 μg of AP-treated RBM did not elicit any signal on phosphospecific antibody immunoblots (see Fig. 1D).
(C) Dephosphorylation of Kv2.1 by brain ischemia induced by CO2 inhalation. RBM prepared from rats treated without (C) or with CO2 gas were size fractionated by SDS-PAGE and analyzed for Kv2.1 phosphorylated at S563 or S603 with corresponding phosphospecific antibodies, or total Kv2.1 with KC antibody. Values in boxes represent the levels of signals as percentages of the control values.
- supplemental material
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Supplemental Figure 2. Heterogeneity of phosphorylation in Kv2.1 clusters in subicular neurons
(A, C) Images of rat subicular neurons double immunofluorescence stained with S563P (A) or S603P (C) antibodies (red) and K89/34 (green) as in Fig. 2B.
(B, D) To quantify relative levels of phosphorylation in individual clusters, staining intensity was measured over a 45 μm segment on the neuronal cell body at the sites indicated by the white lines in the images in (A) and (C). The signal intensity is presented on an arbitrary scale of 0 – 255 (black – white) and plotted against distance (in μm).
