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1.
FIGURE 5.

FIGURE 5. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

Effect on cap-independent translation of N-terminal eIF4GI deletion variants. (A) Structure of eIF4GI deletion variants. (B) Lysates from C were probed with myc-tag and tubulin antibodies. (C) Twenty-four hours after transfection with pcDNA, eIF4GI-e, eIF4GI-Δ1-3, or Ct expression plasmids, HeLa cells were transfected with uncapped c-myc (black bars) or VEGF (black hatched bars) IRES reporters or capped β-globin (gray bars) or uncapped inverse c-myc IRES (gray hatched bars) reporter constructs and lysed 6 h thereafter.

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.
2.
FIGURE 6.

FIGURE 6. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

Interaction between Ct and the c-myc 5′ UTR. (A) RT-PCR of RNA co-IPed with myc-tag antibody. RT-PCR from total cellular RNA, myc-tag IP, or isotype controlled nonspecific mouse IgG from lysed tet-induced Ct-expressing cells previously transfected with the indicated reporters. (B) Effect of GST-Ct or GST alone on radioactively labeled c-myc 5′ UTR RNA in EMSA. Twenty nanograms of labeled c-myc 5′ UTR (105 cpm) were incubated with varying amounts of GST-Ct or GST as indicated. (C) Effect of unlabeled competitors on the interaction between GST-Ct and labeled c-myc 5′ UTR RNA and (D) inverse c-myc 5′ UTR in EMSA. Twenty nanograms of labeled RNA were incubated with GST-Ct and increasing amounts of nonradioactive competitor RNA as indicated. Ratios of competitor RNA were 1:1, 1:2.5, 1:5, and 1:10.

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.
3.
FIGURE 3.

FIGURE 3. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

Dose dependency and specificity of Ct-mediated IRES stimulation. (A) Ct cells were tet-induced from 30 min up to 16 h. At given intervals, uncapped c-myc 5′ UTR (black bars) or m7GTP-capped β-globin leader (gray bars) reporter RNAs were transfected and cells were lysed 6 h thereafter. The lysates were tested by immunoblot with myc-tag or tubulin antibodies (top panel). (B) Ct cells were tet-induced for 16 h and transfected with uncapped reporter RNAs under the control of the indicated sequences: the c-myc- or VEGF- 5′ UTRs; the EMCV-, HCV-, or CBV3 IRESs; the inverted c-myc 5′ UTR. RLuc activity was measured 6 h after transfection and lysates were tested by immunoblot with myc-tag and tubulin antibodies (top panel).

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.
4.
FIGURE 7.

FIGURE 7. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

A Ct mutant with reduced RNA binding capacity fails to stimulate cap-independent translation. (A) Structure of eIF4G, Ct, and the location of two separate elements of a putative RNA recognition motif (Lomakin et al. 2000). Altered amino acids in Mut1 and -2 are indicated by arrows. (B) Co-IP of translation factors with myc-Ct or its variants. Immunoblots of eIF4A, eIF3a, and eIF4G from lysates transfected with the indicated expression plasmids. (C) Stimulation of the indicated 5′ UTRs after transfection of RNA reporters into cells expressing the diverse Ct variants (bars are labeled according to B). (D) EMSA of labeled c-myc 5′ UTR with recombinant GST-Ct or its variants as indicated.

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.
5.
FIGURE 1.

FIGURE 1. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

eIF4G cleavage and tet-inducible myc-eIF4G-b and myc-Ct expressing cell lines. (A) Schematic of eIF4GI structure. Binding domains for PABP, eIF4E, -4A, -3, and Mnk1 are indicated. Numbering refers to amino acids in eIF4GI-a (Byrd et al. 2002). (B) eIF4GI immunoblot of lysates from untreated (−), CBV3-infected (CBV3), or 2Apro transfected (2Apro) HeLa cells. (C) eIF4GI-b and Ct expressing cell lines were tet-induced for 16 h and subjected to immunoblot using eIF4GI, -eIF4E-, eIF4A-, and PABP antibodies. (D) Lysates from eIF4GI-b or Ct expressing cell lines were subjected to cap-sepharose pull down (left panel) or immunoprecipitation (IP, right panel) using anti-myc antibody or mouse IgG control (data not shown). Cap-pull downs were probed with eIF4GI- and eIF4E antibodies, while IPs were probed with eIF4GI-, and eIF4A antibodies. The asterisk denotes a nonspecific background band.

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.
6.
FIGURE 4.

FIGURE 4. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

Effect on translation of eIF4GI-e and -4GI-e variants deficient in binding eIF4E. (A) Structure of eIF4GI-e and -4GI-eEM (mutant amino acids in the eIF4E binding domain are indicated by arrows). (B) Hela cells were transfected with pcDNA, myc-eIF4GI-e, -4GI-eEM, or Ct and lysates analyzed by immunoblot for exogenous myc-tagged proteins, PABP, and eIF4A. (C) m7GTP-cap sepharose binding assays of lysates from B. Lysates were incubated with cap-sepharose, and bound proteins were eluted and probed with eIF4E, eIF4GI, or myc-tag antibodies. Endogenous eIF4E/eIF4G are bound by m7GTP-cap sepharose in all samples. Only wild-type exogenous myc-eIF4G-e associates with the cap. (D) Co-IP assays of lysates from B. IP with myc-tag antibody reveals eIF4A binding to exogenous eIF4G variants in all samples. The asterisk indicates a nonspecific background band. (E) HeLa cells were transfected with c-myc (black bars) and VEGF (black hatched bars) IRES reporters or capped β-globin (gray bars) or uncapped inverse c-myc IRES (gray hatched bars) reporter constructs and lysed 6 h thereafter.

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.
7.
FIGURE 2.

FIGURE 2. From: Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo.

Effect of eIF4G cleavage and inducible eIF4GI-b and Ct expression on c-myc 5′ UTR-mediated translation. (A, left panel) Uncapped c-myc 5′ UTR-driven reporter translation in untreated (−), CBV3-infected (CBV3), 2Apro co-transfected (2Apro) HeLa cells, or stable tet-inducible eIF4GI-b and Ct expressing cell lines. Cells were infected with CBV3 30 min prior to reporter transfection or reporter RNA was cotransfected with 2Apro expression RNA (Dobrikova et al. 2006). Tet induction was initiated 16 h prior to reporter transfection and all cells were lysed 6 h thereafter. (A, right panel) Capped c-myc 5′ UTR reporter translation in tet-inducible eIF4GI-b and Ct expressing cell lines. (B) Structure of c-myc 5′ UTR reporters. All constructs contained the c-myc 5′ UTR. Gray boxes symbolize a stable stem–loop structure followed by a spacer to block scanning (Thoma et al. 2004; Hundsdoerfer et al. 2005). m7G indicates the presence of a cap structure. (C) Fold stimulation of translation upon tet induction of Ct-expressing cells transfected with the indicated reporter RNAs.

Constanze Kaiser, et al. RNA. 2008 October;14(10):2170-2182.

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