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. 2016 Feb 1;27(3):518–534. doi: 10.1091/mbc.E15-07-0504

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© 2016 Williams et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 8: — hnRNP-Q1 represses Gap-43 mRNA translation through the Gap-43 5′-UTR GQ sequence. (A) N2a cells were transfected with 3×Flag-mCherry-tagged GAP-43 reporters with (FL) and without (Δ5′GQ) the 5′GQ ∼56 h after hnRNP-Q1 #1 or Scr siRNA transfection. After ∼16 h, the cells were starved of methionine for 1 h and labeled with the methionine analogue AHA for 2 h. AHA incorporated into newly synthesized proteins was labeled with biotin; 3×Flag-mCherry-tagged GAP-43 was immunoprecipitated; and newly synthesized 3×Flag-mCherry-tagged GAP-43 (predicted to be ∼75 kDa) was visualized by immunoblot with streptavidin and anti-Flag. Top, the streptavidin signal; bottom, total Flag, α-tubulin signal and AHA Flag-GAP-43/Total Flag-GAP-43 merged signals. *, Nonspecific bands. (B) Quantification of AHA Flag-GAP-43 protein levels normalized to total α-tubulin protein levels from 1 or 5% input. n = 6, two-way ANOVA, Holm Sidak’s posthoc, p values: Scr + FL vs. Scr + ΔGQ, p < 0.0001; Scr + FL vs. Q1 + FL, p = 0.0127; Scr + FL vs. Q1 + ΔGQ, p < 0.0001; Scr + ΔGQ vs. Q1 + FL, p < 0.0001; Scr + ΔGQ vs. Q1 + ΔGQ, p = 0.0011; Q1 + FL vs. Q1 + ΔGQ, p < 0.0001. (C) The 3×Flag-mCherry–tagged GAP-43 reporter constructs were overexpressed in N2a cells for ∼16 h, and reporter expression was visualized by Flag immunoblot. n = 5, one-sample t test, p value = 0.0177. (D) N2a cells were transfected with hnRNP-Q1 #1 or Scr siRNA, the 5′ or 5′ΔGQ firefly luciferase construct, and a Renilla luciferase construct for normalization. After 72 h, the cells were trypsinized and processed for luciferase activity. n = 5, two-way ANOVA, Tukey’s posthoc, p values: Scr + 5′ vs. Scr + ΔGQ, p < 0.0001; Scr + 5′ vs. Q1 + 5′, p = 0.0035; Scr + 5′ vs. Q1 + ΔGQ, p < 0.0001; Scr + ΔGQ vs. Q1 + 5′, p < 0.0001; Scr + ΔGQ vs. Q1 + ΔGQ, p = 0.0248; Q1 + 5′ vs. Q1 + ΔGQ, p < 0.0001. (E) N2a cells were transfected with hnRNP-Q1 #1 or Scr siRNA, the 5′GQ or empty vector firefly luciferase construct, and a Renilla luciferase construct for normalization. After 72 h, the cells were trypsinized and processed for luciferase activity. n = 6, two-way ANOVA, Holm Sidak’s posthoc, p values: Scr + E vs. Scr + 5′GQ, p = 0.0010; Scr + E vs. Q1 + E, p = 0.0543; Scr + E vs. Q1 + 5′GQ, p = 0.0694; Scr + 5′GQ vs. Q1 + E, p < 0.0001; Scr + 5′GQ vs. Q1 + 5′GQ, p = 0.0492; Q1 + E vs. Q1 + 5′GQ, p = 0.0014.