Fig 2: The overexpression of PORCN suppressed the surface expression of GluA1, GluA2, and GluA3 in transfected HEK293T cells. (A) Representative images (left) and quantification of the puncta intensity (right) of the surface expression of GluAs in HEK293T cells expressing GluA1 and stargazin (A1), GluA2 and stargazin (A2), or GluA3 and stargazin (A3) and transfected with either PORCN or a control plasmid. (B) Representative images (left) and quantification of the puncta intensity (right) of the total expression of GluAs in HEK293T cells expressing GluA1 and stargazin (B1), GluA2 and stargazin (B2), or GluA3 and stargazin (B3) and transfected with either PORCN or a control plasmid. The white lines in the images represent scale bars (scale bars = 10 μm). All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; ***p < 0.001).

Fig 3: The ATD and CTD of GluAs are not required for the inhibitory effect of PORCN on the surface expression of AMPARs in cultured neurons. (A1,A2) Representative images and quantification of the intensity and density of the surface expression of GluA1 deletion constructs (A1: GluA1-ΔATD, A2: GluA1-ΔC) in cultured neurons expressing GluA1 deletion constructs. (B1,B2) Representative images and quantification of the intensity and density of the surface expression of GluA2 deletion constructs (B1: GluA2-ΔATD, B2: GluA2-ΔC) in cultured neurons expressing GluA2 deletion constructs. (C1–C3) Representative images and quantification of the intensity and density of the surface expression of GluA3 full length and deletion constructs (C1: GluA3-ΔATD, C1: GluA1-Δ824, C3: GluA3-ΔMPR) in cultured neurons expressing GluA3 deletion constructs. The white lines in the images represent scale bars (scale bars = 5 μm). All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig 4: The binding of PORCN to AMPARs is independent of the AMPAR ATD or CTD. (A) Pulldown of GluA1, GluA1-ΔATD, GluA1-Δ824, and GluA1-ΔC expressed in transfected HEK293T cells together with the pulldown of myc-tagged PORCN with an anti-myc antibody. (B) Pulldown of GluA2, GluA2-ΔATD, GluA2-Δ824, and GluA2-ΔC expressed in transfected HEK293T cells together with the pulldown of myc-tagged PORCN with an anti-myc antibody. (C) Pulldown of GluA3, GluA3-ΔATD, GluA3-Δ824, and GluA3-ΔMPR expressed in transfected HEK293T cells together with the pulldown of myc-tagged PORCN with an anti-myc antibody.

Fig 5: EA or GBP alone did not affect ΔFosB and GluR2 expression in cocaine-naïve mice. (A) Timeline for the experimental procedures. (B, C) Cocaine-naïve mice were treated with EA or GBP (1 mg/kg i.p.). Levels of ΔFosB and GluR2 expression were examined by Western blotting (n = 5 per group). The original Western blot images are available in Supplementary Figure S11 and S12, respectively. The bar graphs show the quantification of protein from (B, C). The same control group was used for both Western blotting assays. Abbreviations: Control = Cocaine-naïve mice; EA = cocaine-naïve mice with EA treatment; GBP = Cocaine-naïve mice with i.p. GBP (1 mg/kg) injection.