Figure 8. Sam68 enhances translation of... | Rockefeller University Press

Figure 8.

$Figure 8. Sam68 enhances translation of a reporter gene containing the 3′ UTR of the Spag16 mRNA. (A) HEK293 cells were transfected with a construct expressing GFP-Sam68 in the absence (left) or presence (right) of a construct encoding a constitutively active form of RAS (RASL61Q), which induces cytoplasmic translocation of the protein. The absorbance profile (OD = 254 nm) of sucrose gradient sedimentation of cells extracts from control (Ctrl) or RAS-transfected (RAS) cells are shown. The bottom panels show the Western blot analysis of Sam68 distribution in each fraction of the gradients. Fractions were also analyzed for ERK2 and for phosphoERKs (pERKs) to determine the activation of mitogen-activated kinases downstream of RAS. (B) Luciferase reporter assay of the effect of wild-type or mutated Sam68 (Sam68m1) on the expression of luciferase in the presence or absence of the 3′ UTR of Spag16 mRNA. HEK293 cells were cotransfected with empty vector, mycSam68, or mycSam68m1 and a constitutively active form of RAS to induce cytoplasmic translocation and polysome association of Sam68. A schematic representation of the Firefly luciferase constructs used is shown above the graph. Western blot analysis of the expression of wild-type and mutant mycSam68 is shown. Western blotting for ERK2 was performed as a loading control. The data of the reporter assay are expressed as the mean ± SD of three experiments and represented as ratios between Firefly and Renilla luciferase activity in each sample. (C) Extracts from HEK293 cells transfected with the Luciferase-Spag16 reporter gene and RAS alone or in combination with wild-type or mutant Sam68 were separated on sucrose gradients, then RNA was isolated from each fraction and pools of the polysomal (1–5) or RNP (8–10) fractions were used for real-time PCR as described in Fig. 6 B using Gapdh as the internal standard.$

Sam68 enhances translation of a reporter gene containing the 3′ UTR of the Spag16 mRNA. (A) HEK293 cells were transfected with a construct expressing GFP-Sam68 in the absence (left) or presence (right) of a construct encoding a constitutively active form of RAS (RASL61Q), which induces cytoplasmic translocation of the protein. The absorbance profile (OD = 254 nm) of sucrose gradient sedimentation of cells extracts from control (Ctrl) or RAS-transfected (RAS) cells are shown. The bottom panels show the Western blot analysis of Sam68 distribution in each fraction of the gradients. Fractions were also analyzed for ERK2 and for phosphoERKs (pERKs) to determine the activation of mitogen-activated kinases downstream of RAS. (B) Luciferase reporter assay of the effect of wild-type or mutated Sam68 (Sam68m1) on the expression of luciferase in the presence or absence of the 3′ UTR of Spag16 mRNA. HEK293 cells were cotransfected with empty vector, mycSam68, or mycSam68m1 and a constitutively active form of RAS to induce cytoplasmic translocation and polysome association of Sam68. A schematic representation of the Firefly luciferase constructs used is shown above the graph. Western blot analysis of the expression of wild-type and mutant mycSam68 is shown. Western blotting for ERK2 was performed as a loading control. The data of the reporter assay are expressed as the mean ± SD of three experiments and represented as ratios between Firefly and Renilla luciferase activity in each sample. (C) Extracts from HEK293 cells transfected with the Luciferase-Spag16 reporter gene and RAS alone or in combination with wild-type or mutant Sam68 were separated on sucrose gradients, then RNA was isolated from each fraction and pools of the polysomal (1–5) or RNP (8–10) fractions were used for real-time PCR as described in Fig. 6 B using Gapdh as the internal standard.