Figure 2. The 5-HT₃A(C312A) mutation does not disrupt pentamer formation or receptor internalization.

A, HEK293 cells transiently expressing Myc-tagged 5-HT₃A (lane 1 and 4), its mutant C312A (lane 2 and 5), 5-HT₃B (lane 3) or GFP (lane 6) were homogenized and membrane proteins were solubilized in RIPA buffer. The extracted membrane protein was subjected to Western blot analysis in the presence or absence of DTT. The positions of molecular mass markers (in kDa) are indicated on the left. Two additional experiments gave similar results. B, for total labelling HEK293 cells, transiently expressing HA-5-HT₃A or mutant Myc-5-HT₃A(C312A) receptors, were fixed, permeabilized and then exposed to anti-HA or Myc antibodies. To examine receptor trafficking, cells were first surface-labelled at room temperature with anti-HA or anti-Myc antibodies. Subsequently, cells were either fixed immediately (control) or incubated at 37°C for 30 min before fixation. Bound primary antibodies were detected with Alexa Fluor 488 (rendered in green) or 594 (rendered in red) conjugated secondary antibodies, and examined by confocal microscopy. Representative images from two independent experiments performed in duplicate are shown. Scale bar is 16 μm.