Journal: International journal of molecular sciences

Article Title: Reovirus μ2 Protein Impairs Translation to Reduce U5 snRNP Protein Levels.

doi: 10.3390/ijms24010727

Figure Lengend Snippet: Figure 2. The µ2 protein impairs translation: (A) WB of newly synthesized proteins in HEK 293T cells expressing GFP, µ2-GFP, or GFP- µ2. Cells were transfected for 48 h, newly synthesized proteins were metabolically labeled with AHA for 4 h (or DMSO for control), a biotin moiety was added using CLICK chemistry and revealed using streptavidin-HRP. A loading control (actin) was loaded and a WB against GFP was realized to validate the expression of GFP and fusion protein between GFP and µ2; (B) quantification of three independent experiments. Biological replicates, n = 3, one-way ANOVA with Dunnett’s multiple comparisons test against the GFP alone condition (***, p ≤0.001); (C) polysome profiles for control GFP or µ2-expressing cells. Polysome to monosome ratio (P/M) and heavy to light ratio (H/L) were calculated as described in the material and methods section. Respective profiles were overlapped by aligning the 40S to the same height; (D) polysome to monosome ratio (P/M) and heavy to light ratio (H/L) from three independent experiments for control GFP or µ2-expressing cells. Biological replicates, n = 3, one-way ANOVA with Dunnett’s multiple comparisons test against the GFP alone condition (***, p ≤0.001); (E) relative mRNA level determined by qPCR for EFTUD2, PRPF8, SNRNP200, and GAPDH in 40S + 60S, 80S, and polysomal (P1 + P2 + P3) fractions from GFP or µ2-GFP expressing cells at 48 h post-transfection. Lysates were prepared, separated on a 5–50% sucrose gradient, and 30% of each fraction was subjected to RNA extraction using Qiazol. RNA was reverse transcribed using a fixed volume for each sample and subjected to qPCR with U6 snRNA as the housekeeping gene, as it was stable between all fractions. The relative expression is calculated against the first sample in the GFP condition for the 40S + 60S fraction. n = 3, biological replicates, two-way ANOVA with Dunnett’s multiple comparisons test against the GFP control for each fraction (ns, p > 0.05); and (F) WB against GFP, RPL19 (large subunit), RPS2 (small subunit), and actin in polysomal fractions from control GFP or µ2-expressing cells at 48 h post-transfection in HEK 293T cells. Results presented are mean ± standard deviation.

Article Snippet: The antibodies used in this study are the following: Actin (Sigma, A5441, 1:10,000), EFTUD2 (Abcam, ab188327, 1:2000), FLAG (Sigma, F1804, 1:1000), GFP (Santa Cruz Biotechnology, sc-9996, 1:8000), PRPF8 (Abcam, ab79237, 1:1000), Streptavidin-HRP (ThermoFisher Scientific, N100, 1:5000), RPL19 (Santa Cruz Biotechnology, 1:1000), RPS2 (1:2000, a kind gift from the Mark Bedford lab), Vinculin (Santa Cruz Biotechnology, sc-73614, 1:1000).

Techniques: Synthesized, Expressing, Transfection, Metabolic Labelling, Labeling, Control, RNA Extraction, Reverse Transcription, Standard Deviation