Fig. 4.

CaMKIIβ-induced downregulation of cell surface GABA_B receptors depends on Lys-63-linked ubiquitination. Neurons were transfected with either EGFP (control), CaMKIIβ, or CaMKIIβ plus ubiquitin (Ub) or one of its mutants (Ub(K63): only Lys-63 linkages, Ub(K63R): no Lys-63 linkages, Ub(K48R): no Lys-48 linkages, Ub(KO): no polyubiquitin linkages (only mono-ubiquitination). Two days after transfection, neurons were stained for GABA_B receptors using GABA_B2 antibodies. Only interference with K63-linked ubiquitination prevented CaMKIIβ-mediated downregulation of the receptors. Representative images of stained neuronal somata (scale bar, 5 μm). The scatter plot shows quantification of fluorescence intensities. The fluorescence signal of neurons transfected with EGFP was set to 100%. The data represent the mean ± SD of 28–30 neurons from two independent experiments. ***p < 0.001, ^nsp > 0.05; one-way ANOVA, Dunnett’s multiple comparison test