Figure 7. ERK phosphorylation of M2 and M4 mAChRs was not affected by altering the mode of internalization.

(A,B) Phosphorylation of ERK1/2 was determined by western blotting for HEK293 cells transfected with pcDNA3.1 empty vector, EGFP-tagged wild type and deletion mutants of M2 (A) or M4 (B) after treatment with 100 μM CCh for 5 min. (C,D) Time courses of phosphorylation of ERK1/2 were determined by western blotting for HEK293 cells transfected with EGFP-tagged M2 and M2-based chimeric M2/M4(R³⁸⁶-A³⁹³) (C) or M4 and M4-based chimeric M4/M2(K³⁷⁴-S³⁸⁰) (D). Cells were treated with 100 μM CCh for indicated time points as depicted. Total ERK was used as a loading control. (E) The densitometric analysis of ERK 1/2 phosphorylation elicited by the activated receptor depicted in C and D was normalized to the maximum value and plotted. Mean ± SEM of immunoblots in three independent experiments are shown.