Journal: Brain Pathology
Article Title: Targeting the interaction of GABA B receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices
doi: 10.1111/bpa.13099
Figure Lengend Snippet: R1‐Pep treatment reduced aberrant expression of K ir 3.2 channels after glutamate stress. Glutamate‐stressed neurons were treated for 16 h with R1‐Pep (10 μg/ml) or Ctrl‐Pep (10 μg/ml) and then stained with K ir 3.2 antibodies. (A) Immunofluorescence staining: top, representative images (scale bar: 5 μm); bottom, quantification of fluorescence intensities. N = 30 neurons per condition from two independent experiments. Signals were normalized to no Pep control. Two‐way ANOVA with Tukey's multiple comparison test (ns, p > 0.05; ****, p < 0.0001). (B) Western blotting: top panel, staining for K ir 3.2 channels; bottom panel, staining for total protein used for normalization. Control, untreated cultures; Glu, glutamate‐stressed cultures; Glu + R1‐Pep, glutamate‐stressed cultures treated with R1‐Pep; Glu + R1‐Pep + CGP, glutamate‐stressed cultures treated with R1‐Pep and the GABA B receptor antagonist CGP 56999. Signals were normalized to control. N = 11 cultures per condition from four independent neuron preparations. One‐way ANOVA with Tukey's multiple comparison test (ns, p > 0.05; *, p < 0.05; **, p < 0.01 ***, p < 0.0005).
Article Snippet: Mouse Akt (1:250 for immunofluorescence [IF], Cell Signaling Technology #2920), rabbit pAkt‐S473 (1:1000 for IF, Cell Signaling Technology #4060), rabbit CaMKII (1:1000 for IF, 1:100 for PLA; Abcam #ab52476), mouse CaMKIIβ (1:1500 for Western blotting [WB], Thermo Fisher Scientific #13‐9800), rabbit phospho‐CaMKII(Thr286) (1:500 for WB, Cell Signaling Technology #12716), rabbit Ca V 2.2 (1:1000 for IF and 1:200 for Western blotting; Alomone Labs #ACC‐002), mouse GABA B1 (1:250 for IF and Western blotting, 1:100 for PLA; Abcam #ab55051), rabbit GABA B1b directed against the N‐terminus of GABA B1b (affinity‐purified, 1:100 for IF; custom made by GenScript) [ ], rabbit GABA B2 directed against the N‐terminus of GABA B2 (affinity‐purified, used for cell surface staining, 1:25 for IF; custom made by GenScript) [ ], rabbit GABA B2 (1:500 for IF, 1:100 for PLA, 1:800 for Western blotting; Abcam #ab75838), mouse GFP antibody (for immunoprecipitation, TaKaRa #632381), rabbit K ir 3.2 (1:250 for IF and Western blotting, Alomone Labs #APC‐006), mouse GSK3β (1:250 for IF, Abcam #ab93926), rabbit pGSK3β‐S9 (1:1500 for IF, Abcam #ab131097), rabbit pGSK3β‐Y216 (1:1500 for IF, Abcam #ab75745), rabbit ubiquitin K63‐specific (clone Apu3, 1:50 for PLA; Millipore #05‐1308), rabbit NeuN (1:400 for IF; Millipore #ABN78), mouse phospho‐serine (1:150 for PLA, Sigma‐Aldrich #P5747), Alexa Fluor 647 and 488‐conjugated streptavidin (1:100; Jackson ImmunoResearch Laboratories #016‐600‐084 and #016‐540‐084).
Techniques: Expressing, Staining, Immunofluorescence, Fluorescence, Western Blot
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