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. 2003 Jun;121(6):495–510. doi: 10.1085/jgp.200208770

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Copyright © 2003, The Rockefeller University Press

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Figure 9. — Western blots assaying Gβ expression. (A) Blots shown are for N-type calcium channel (α_1B/β_2a/α₂δ) expressed alone, or coexpressed with wild-type or mutant Gβ₁. Samples were obtained from the membrane fraction of cells. The soluble fraction gave far weaker signals. The first five lanes contain varying levels of purified, bovine Gβ₁ to demonstrate that samples are in the linear range. “HEK” is HEK 293 cell lysates that have undergone calcium phosphate transfection using only cDNA encoding T antigen. “Channel” identifies extracts from cells transfected with only channel subunits. All other lanes are N-type channel subunits expressed with Gγ₂ and either wild-type or mutant Gβ₁, as labeled. Mutations D186A and D228A traveled slightly faster than other Gβs expressed, due to the change in charge that occurs with alanine mutation. All lanes were quantified based on band density and compared with the controls. Lanes with overexpressed Gβ (wild-type or mutant) averaged 9.15 ng with a standard deviation of 2.34. (B) Blots for wild-type and mutant Gβ₁ coexpressed with P/Q-type calcium channels. Again, various levels of purified Gβ₁ are shown in the first five lanes. The “channel” lane shows lysates from cells transfected with only the P/Q-type channel subunits (α_1A/β_2a/α₂δ). Lanes with overexpressed Gβ (wild-type or mutant) averaged 10.63 ng with a standard deviation of 1.56. (C) Solid curves reproduce fits for modulation of N-type channels by wild-type Gβγ, taken from Fig. 2, B₁ and B₂. Dashed lines represent the simulated changes that would occur when G-protein concentration is increased or decreased by a factor of two, holding all other parameters fixed. (D) Analogous sensitivity analysis for modulation of P/Q-type channels by wild-type Gβγ, with solid curve fits reproduced from Fig. 3, D₁ and D₂, and format identical to that in C. Again, dashed lines represent the simulated changes that would occur when G-protein concentration is increased or decreased by a factor of two, holding all other parameters constant.