Fig. 4
Gβ selectivity of Nb5. a Multiple sequence alignment of key amino acid residues of Gβ that interact with Nb5. *The amino acid numbering of Gβ5 has an off-set of −50. b Nb5-mediated Gβγ extraction from mouse brain. Solubilized and partially purified extracts from mouse brain (left) were subjected to either Nb5- (right, lanes 1 and 2) or Nb17- (right, lanes 3 and 4) mediated immobilized-metal affinity purification of Gβγ. c In-gel protein digestion of Gβ subtypes (band marked in b with asterisks) purified from mouse brain. Peptides were separated, analyzed and searched against a full mouse proteome to identify unique peptides from Gβ1, Gβ2, Gβ3, and Gβ4. d Schematic diagram of the effect of Gβγ-binding proteins on the BRET assay. Co-transfection of HEK293T/17 cells with Venus-Gβγ and masGRK3ct-Nluc-HA produced a high BRET signal through their direct interaction (left). Introduction of Gβγ-binding proteins (e.g., the Gα subunit) competed with masGRK3ct-Nluc-HA, lowering the BRET signal (right). e Effects of Nb5 on the interaction of Gβγ and the C-terminus of GRK3. The maximum BRET signal was determined by co-transfection of different Venus-Gβ subtypes + Gγ2 pairs and masGRK3ct-Nluc-HA (grey). A minimum BRET signal also was determined after co-transfection of Venus-Gβ1γ2 and masGRK3ct-Nluc-HA with an excess amount of GαoA (pink). Effects of Nb5 and Nb17 were examined by co-transfection of Venus-Gβγ and masGRK3ct-Nluc-HA with either Nb5 (greencyan) or Nb17 (purple). Experiments were performed with Gβ subtypes 1–4 + Gγ2 pairs. Each bar represents the mean of six replicates. Similar results were obtained in three independent experiments. Results are expressed as the mean ± SEM. One-way ANOVA with Tukey’s post hoc multiple comparison test relative to the Gβ1γ2/GRK3ct control, ***P ≤ 0.001, n = 6 replicates. f Western blot quantification of the expression levels of Gβ1–4, GRK3, Gα, Nb5, and Nb17 (Full blots are shown in Supplementary Figure 4a)
