Figure 2.

Knockdown of clathrin heavy chain hinders M₂ receptor internalization. (A,B) HEK 293T cells were depleted of CHC by two different siRNAs and transfected with M₂-EGFP. Cells were starved for 18 h in serum-free medium and either left untreated or stimulated with 1 mM CCh for 15 min. Cells were fixed and immunostained for CHC, and the nuclei were stained with DAPI (shown in blue). Scale bars: 10 μm. Single cells still expressing some CHC are marked with an asterisk; (C) The number of cells displaying M₂ receptor internalization was quantified and is shown as the percentage of cells showing intracellular M₂ localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: *** p < 0.001; (D) Equal amounts of protein in cell lysates were separated by SDS-PAGE and immunoblotted to monitor CHC knockdown efficiency (about 60%).