Figure 5: The PX-RICS-dependent trafficking complex is required for surface expression of GABAARs.
(a) Mouse cortical neurons (10 DIV) were transfected with the indicated siRNA (10 nM) plus Red Fluorescent Oligo (10 nM). Surface (green) and intracellular (blue) levels of the γ2 subunit in siRNA-introduced neurons (red) were analysed by surface labelling. The settings of the microscope, lasers and detectors were kept constant during image acquisition. Scale bars, 10 μm. (b) Quantitative analysis of surface-expressed and intracellular γ2. n=10 (images in each siRNA). (c) PX-RICS−/− cortical neurons (14 DIV) were transfected with plasmids expressing FLAG-tagged wild-type (WT) or mutant PX-RICS as indicated. ΔRSKSDP, a mutant lacking the 14-3-3-binding motif (amino acids 1,793–1,798); S1796A, a mutant in which Ser-1796, the CaMKII phosphorylation site in the 14-3-3 binding motif, is replaced with Ala; ΔGBR, a mutant lacking the GABARAP-binding region (amino acids 562–796). Surface (green) and intracellular (blue) levels of the γ2 subunit in transfected neurons (red) were analysed by surface labelling. The settings of the microscope, lasers and detectors were kept constant during image acquisition. Scale bars, 10 μm. (d–g) Quantitative analyses of surface-expressed γ2 in entire neurons (d) and the representative dendrites (e). The number (f) and averaged size (g) of surface-expressed γ2 puncta within 40-μm portion of the representative dendrites are also quantified using ImageJ. n=10 (images in each construct). Data are represented as means±s.e.m. NS, not significant; ***P<0.001 (unpaired two-tailed Student’s t-test, compared with siControl (b) or vector control (d–g)).
