Effects of Tra2β over-expression and silence on RGS4 expression in SH-SY5Y cultured cells and rat LC.
(A) An illustration of GFP-RGS4 minigene construction. The GFP reporter gene was fused in-frame with RGS4 genomic DNA sequences consisting of RGS4 exons 4, 5, 6 and 7. Exons are shown as boxes, introns as lines. (B) Tra2β over-expression increased the ratio of RGS4-1 isoform to RGS4-4. SH-SY5Y cells were co-transfected with the GFP-RGS4 minigene plus Tra2β over-expression plasmids (Tra2β) or empty vectors (Ctrl). The GFP-RGS4-1 (43 kDa) and GFP-RGS4-4 (38 kDa) proteins were detected by GFP antibody. The structures of the GFP-RGS4-1 and GFP-RGS4-4 variants were illustrated on the left. (C) Tra2β over-expression increased relative mRNA level (%GAPDH) of RGS4-1 isoform (4.26±0.77), but not RGS4-4 isoform (1.06±0.07, p = 0.67) in SH-SY5Y cells. (D) Tra2β RNAi reduced relative mRNA level (%GAPDH) of RGS4-1 isoforms (0.65±0.08), but not RGS4-4 isoforms (1.14±0.07, p = 0.15) in SH-SY5Y cells. (E) Relative protein level (%GAPDH) of RGS4 in SH-SY5Y cells transfected with increasing amounts of Tra2β over-expression plasmids. Numbers indicate the microgram of Tra2β over-expression plasmids used for transfection. *p<0.05, **p<0.01, ***p<0.001 compared to 0 µg group, using one-way ANOVA. (F) Tra2β RNAi decreased relative protein level (%GAPDH) of RGS4 (0.55±0.03) in SH-SY5Y cells. (G) Illustration of rat right LC infected with lentivirus expressing siRNAs against Tra2β (si-Tra2β LV), and left LC infected with negative control lentivirus (NC LV) under fluorescent microscope. (H) Infection of si-Tra2β LV reduced Tra2β (0.57±0.06) and RGS4 (0.51±0.04) protein levels (%GAPDH) in rat LC. (I) Infection of si-Tra2β LV reduced Tra2β (0.56±0.01) and RGS4 (0.59±0.07) protein levels (%GAPDH) in PC12 cells. The molecular weight of Tra2β, RGS4 and GAPDH are 40 kDa, 28 kDa and 38 kDa, respectively. Data are expressed as mean ± s.e.m. from at least three independent experiments; “n” as denoted inside bar graphs, represents the number of cultures, except for (H) where they represent the number of rats. *p<0.05, **p<0.01, ***p<0.001, using unpaired two-tailed Student’s t-tests for real-time qPCR experiments and paired t-tests for immunoblots.