Figure 2
From: Alternative production of pro-death Bax∆2 protein via ribosomal frameshift in Alzheimer’s disease
Ribosomal frameshift serves as an alternative mechanism for Bax∆2 protein production. (a) Diagram indicating the possible outcomes of dual-luciferase assay with or without frameshift. E1-3, Bax∆2 exon 1 and 3 with G8 microsatellite; R luc, Renilla luciferase; F luc, firefly luciferase. (b) Dual-luciferase activities from in vitro transcribed/translated products from (a); controls: E4, Bax∆2 exon 4 (out of frame); Vec., empty dual-luciferase vector (out of frame). Statistical significance was calculated with one way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent standard deviation (SD). ****, p < 0.0001. (c) Diagram of reading frameshift assay with two-tagged constructs (N-terminal Flag tag, green, and C-terminal HA tag, red). E3-5, Bax∆2 exon 3 to 5. (d) Immunofluorescence staining of HT22 cells transfected with the two-tagged constructs indicated in (b). Scale bar, 10 mm. (e)Diagram of constructs and antibodies used to detect reading frameshift through immunoblot. Antibody recognition sites were labeled on the top. (f) Immunoblotting of HT22 cells transfected with antibodies indicated; Original blots are shown in Supplementary Fig. 3; (g) quantitation of the intensity (grey scale) of bands from three independent HA immunoblots. The percentage of read-through was calculated with G7 control as 100%. Error bars represent SD. The quantitation of the bands for 2D4 antibody is shown in Supplementary Fig. 3e.
