anti dap5 antibody  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) TCGA cohort for metastasis-free survival for ER − /PR − /Her2 − (triple-negative) breast cancer (TNBC) patients. Kaplan-Meier estimates derived from mRNA expression data on the basis of high and low expression levels of eIF4G2 mRNA (top) and eIF3d (bottom). Univariate analysis was performed using the log rank test. Cox proportional hazard regression models were also used. (B) Representative IHC staining showing score for DAP5 protein levels of TNBC primary tumors that did not metastasize during 8-year follow up, and primary tumor and recurrent matched metastases that recurred within 8 years. See also and for IHC scoring summaries, statistical analysis, patient demographics, and tumor characteristics. Patient demographics are representative of first clinical presentation for the overall U.S. TNBC population.

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Derivative Assay, Expressing, Immunohistochemistry

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) Representative immunoblots of 3 independent studies of ITG proteins in Nsi and DAP5-silenced MB-231 cells. Dox induction of shRNAs for 4 days followed by immunoblot analysis of equal protein amounts. (B) Representative immunoblots of 3 independent studies of EMT biomarker protein levels in Nsi and DAP5-silenced 4T1 and MB-231 cells carried out as in (A). (C) Representative immunoblots of 3 independent studies of eIF4E and eIF2α proteins in Nsi and DAP5-silenced MB-231 cells carried out as in (A). (D) Representative immunoblots of 3 independent studies to test requirement for eIF3d cap binding activity for DAP5-dependent mRNAs in MB-231 cells. Cells were small interfering RNA (siRNA) silenced for eIF3d for 1 day and transfected with vectors expressing WT, α5, or α11 cap binding mutants of eIF3d for 2 days, and equal protein amounts analyzed using immunoblot. (E) Matrigel Transwell invasion assays performed with 4T1 and MB-231 cells silenced for 24 h with Nsi or DAP5 Dox-inducible shRNAs. Mean number of invading cells/field with SEM from 3 replicates for each cell line. See also . (F) Cell migration wound healing assays performed with 4T1 and MB-231 cells silenced for 24 h with Nsi or DAP5 Dox-inducible shRNAs. Time 0 represents 100% wound separation of the cell layer. Mean of percentage of migrated cell surface areas (percentage closure) with SEM from 3 replicates for each cell line. See also . (G) Induction of apoptosis by cell detachment (anoikis) determined in 4T1 and MB-231 cells silenced for 24 and 48 h for Nsi, DAP5, or eIF4GI, comparing cells maintained on adherent or ultra- low-adherence plates. Percentage cell apoptosis determined by annexin V-fluorescein isothiocyanate (FITC) staining and flow cytometry, quantified using FlowJo software. Mean with SEM of 3 independent studies per cell line. (H) Representative immunoblot of 3 independent studies of DAP5, eIF4G1, eIF3d, and control β-actin protein levels in Nsi or DAP5-silenced 4T1 and MB-231 cells, silenced for 2 days with Dox-inducible shRNAs. Data are represented as mean ± SEM. n.s., not significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired parametric two-tailed t test.

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Western Blot, Biomarker Assay, Binding Assay, Activity Assay, Small Interfering RNA, Transfection, Expressing, Migration, Staining, Flow Cytometry, Software, Two Tailed Test

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) NOD/SCID/γ mice orthotopically injected with 5 × 10 6 MB-231 cells stably transformed to express Firefly luciferase and red fluorescent protein (RFP), Dox-inducible Nsi or shDAP5 RNAs. Silencing initiated by Dox addition to drinking water 28 days after tumor cell implantation, tumors ~100–150 mm 3 in size. At 49 days, mice were sacrificed, tumors and lungs collected. (B) Growth of tumors was recorded every 7 days by quantitative caliper measurement. n = 8 or 9 mice per group repeated twice. Volumes of non-silenced control compared with DAP5-silenced tumors was not statistically significant. (C) Primary tumors weighed at excision 49 days. Non-silenced control compared with DAP5-silenced tumors not statistically significant. n = 7–9 mice/group. Representative of 2 trials. (D) Equal protein amounts of whole tumor lysates at 49 days post-excision, subjected to immunoblot analysis for DAP5 and control actin levels. n = 4 tumors (of 8 or 9 mice) per arm were evaluated. (E) Representative Firefly bioluminescent images from 8 or 9 mice per group (3 shown), silenced for Nsi or DAP5 as in (A) immediately prior to sacrifice at 49 days. Repeated 3 times. (F) Lungs excised, subjected to bioluminescent imaging for RFP. Representative images of 8 or 9 mice per group (4 shown) of mice silenced for Nsi or DAP5 as in (A). Repeated twice. (G) Quantification of metastatic burden in lungs of mice silenced for Nsi or DAP5 as in (A). Total RFP fluorescent flux quantified (photons [ps]/sec), representative of total lung tumor burden, at 49 days. n = 8 or 9 mice per group. Repeated twice. (H) Representative H&E-stained images of lungs of 8 or 9 mice per group harvested at 49 days. Arrows indicate dark staining tumor masses. (I) Quantification of number of lung metastases (mets) per field at 1003 magnification of H&E-stained lungs as shown in (H). A minimum of 5 fields per lung were quantified from 9 mice/group, mean plus SEM. (J) DAP5-silenced tumors have reduced levels of EMT proteins. Immunoblot of equal protein amounts from control Nsi and shDAP5-silenced tumors harvested at 49 days. Representative results of 6 tumors are shown. Data are mean plus SEM. Statistical significance was assessed by two-way ANOVA with Dunnett post-ANOVA test determination for analysis of repeated measures. n.s., not significant. ***p < 0.001. See also and .

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Injection, Stable Transfection, Transformation Assay, Luciferase, Western Blot, Imaging, Staining

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) Schema of animal study. Balb/c mice subcutaneously injected in the flank with 5 × 10 5 4T1 cells stably transformed to express Firefly luciferase and red fluorescent protein (RFP), Dox-inducible Nsi or shDAP5 RNAs. Silencing initiated 8 days after tumor cell implantation, tumors at 75–100 mm 3 in size, by Dox addition to drinking water. At 20 days, mice were sacrificed and tumors and lungs collected. (B) Equal protein amounts of whole tumor lysates at 20 days post-excision subjected to immunoblot analysis for DAP5 protein and control actin levels. n = 4 tumors per condition were evaluated. (C) mRNA extracted from tumors removed at 20 days and equal RNA amounts used to determine eIF4G2 mRNA levels normalized to Gapdh mRNA. n = 3 tumors per condition; mean and SEM are shown. (D) Growth of tumors recorded every 2–4 days by quantitative caliper measurement. n = 8 or 9 mice per group repeated twice. Volumes of Nsi control compared with DAP5-silenced tumors not statistically significant. (E) Representative Firefly bioluminescent images of 8 mice per group (4 shown), silenced for Nsi or DAP5 as in (A), immediately prior to sacrifice at 20 days. Repeated 3 times. (F) Lungs excised from mice and subjected to fluorescence imaging for RFP. Representative images of 8 or 9 mice per group (4 shown) of mice silenced for Nsi or DAP5 as in (A). Repeated twice. (G) Quantification of metastatic burden in lungs of mice silenced for Nsi or DAP5 as in (A). Total RFP fluorescence flux quantified (photons [ps]/sec), representative of total lung tumor burden harvested at 20 days. n = 7 or 8 mice per group. Repeated twice. (H) Quantification of eIF4G2 mRNA levels in Nsi and DAP5-silenced metastases in lungs of mice. n = 3 per group. Lungs excised, tumor cells dispersed, isolated by RFP FACS, subjected to qRT-PCR for eIF4G2 mRNA normalized to Gapdh mRNA. (I) Representative H&E-stained images of lungs of 8 mice/group harvested from mice bearing Nsi or shDAP5 tumors. Arrows indicate dark staining tumor masses. (J) Quantification of number of lung metastases (mets) per field at 100× magnification of H&E-stained lungs as shown in (I). A minimum of 5 fields per lung quantified, mean plus SEM of n = 8 mice per condition. Data are mean plus SEM. Statistical significance was assessed by two-tailed Student’s t test for unpaired experimental values (C and H), or two-way ANOVA with Dunnett post-ANOVA test determination for analysis of repeated measures with (D, G, and J). n.s., not significant. ***p < 0.001. See also .

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Injection, Stable Transfection, Transformation Assay, Luciferase, Western Blot, Fluorescence, Imaging, Isolation, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) MB-231 cells (1 × 10 7 ) expressing RFP and Dox-inducible control Nsi or shRNA to DAP5 implanted in mammary fat pad of NOD/SCID/γ mice, Dox induced at 28 days, when tumors 100–150 mm 3 in size, maintained for 20 days, tumors with stroma excised, embedded, sectioned for immunofluorescence microscopy for RFP (red) and DAPI (blue) stained nuclei. Representative images of RFP expressing MB-231 cells beyond the periphery of tumor, two different tumors analyzed, 3 fields each. Scale bar, 200 μm. Arrows indicate direction of tumor cell stromal migration away from tumor capsule. (B) Quantitation of images in (A). Quantitation determined from 2 independent tumors, 3 fields each (n = 6), using ImageJ software. (C) Tumors obtained as in (A) from non-silenced Nsi control and DAP5-silenced tumor specimens sectioned, IHC stained for CD31 to highlight tumor vasculature. Representative sections shown from 5 different tumors. Scale bar, 1,000 μm. (D) Quantitation of CD31 stained MB-231 cells from images in (C). Quantitation determined from 3 different tumors, 3 fields each. (E) Schema of animal study for survival determination with DAP5 tumor-specific silencing. Animals subcutaneously injected in flank with 1 × 10 5 4T1-Nsi or 4T1-shDAP5 cells. At 12 days, Dox added to the drinking water to induce eIF4G2 mRNA silencing. (F) Tumor growth recorded every 2–4 days by quantitative caliper measurement until 22 days as control mice began to die. n = 8–10 mice per group, repeated twice. (G) Survival of mice with 4T1-Nsi or 4T1-shDAP5 tumors silenced starting at 13 days. n = 15 mice/group. Animals either died or were sacrificed when terminally moribund. Statistical significance was determined using unpaired non-parametric log rank Mantel-Cox test. Data are mean with SEM. Statistical significance (B, D, and F) assessed using two-tailed Student’s t test.

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Expressing, shRNA, Immunofluorescence, Microscopy, Staining, Migration, Quantitation Assay, Software, Injection, Two Tailed Test

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) Schema of animal study. Balb/c mice subcutaneously injected in the flank with 1 × 10 7 4T1 cells stably transformed to express Firefly luciferase and RFP, Dox-inducible Nsi or shDAP5 RNAs. Tumors aseptically excised at 12 days, ~100–125 mm 3 in size, Dox added to drinking water at 13 days to express shRNAs. At 23 days, mice were sacrificed, and lungs were collected. (B) Primary tumors weighed at excision at 12 days, weight of non-silenced control compared with DAP5-silenced tumors. n = 10–13 mice/group. (C) Growth of tumors recorded 6 and 12 days prior to surgical excision of tumors, by quantitative caliper measurement. n = 8 mice per group repeated twice. (D) Lungs excised from 2 mice per group at 12 days prior to shRNA induction and subjected to fluorescent imaging for RFP. Images show qualitatively similar metastatic tumor burden in lungs of mice prior to Nsi or DAP5 silencing. (E) Lungs excised from mice at 23 days, subjected to fluorescent imaging for RFP. Representative images of 8 mice per group (4 shown) for mice silenced for Nsi or DAP5 as in (A). n = 8 mice/group. Repeated twice. (F) Quantification of metastatic burden in lungs at 23 days of mice silenced for Nsi or DAP5 as in (A). Total RFP fluorescence flux quantified (photons [ps]/sec), representative of total lung tumor burden. n = 8–10 mice per group. Repeated twice. (G) Quantification of number of lung metastases (mets) per field at 100× magnification of H&E-stained lungs at 31 days. A minimum of 5 fields per lung quantified, mean plus SEM. n = 11 or 12 mice per condition. (H) Quantification of eIF4G2 mRNA levels in Nsi and DAP5-silenced metastases in lungs of mice at 31 days. n = 3 per group. Lungs excised, tumor cells dispersed, isolated by FACS gating on RFP and subjected to qRT-PCR for eIF4G2 mRNA normalized to Gapdh mRNA. Data are mean plus SEM. Statistical significance by two-way ANOVA with Dunnett post-ANOVA test determination for analysis of repeated measures. n.s., not significant. **p < 0.01 and ***p < 0.001.

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Injection, Stable Transfection, Transformation Assay, Luciferase, shRNA, Imaging, Fluorescence, Staining, Isolation, Quantitative RT-PCR

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: (A) Representative immunoblot of select translation factor proteins in equal amounts of lysates from less transformed parental MB-231 cells, and two increasingly more transformed and more metastatic variants, LM0 and ML2 cells. (B) Representative immunoblot of 3 independent studies of parental MB-231 cells and parental MB-231 cells stably transfected with DAP5 cDNA (MB-231 OE cells). (C) Representative light field microscopic images of trypan blue stained parental MB-231 cells and MB-231 OE cells. n = 3 independent plating of cells, images representative from 12 different fields chosen at random. (D) Matrigel Transwell invasion assays performed with parental MB-231 cells or MB-231 OE cells, carried out as in . Results represent the mean of invading cells/field with SEM from 3 independent studies. Statistical analysis by unpaired two-tailed t test. (E) Cell migration wound healing assay. performed with parental MB-231 cells and MB-231 OE cells carried out as in . Statistical analysis by unpaired two-tailed t test. (F) Mice were injected in the retro-orbital (RO) sinus with 10 3 parental MB-231 cells or MB-231 OE cells, both expressing Firefly luciferase. Ten days later representative Firefly bioluminescent images were obtained from 3–5 mice per group (3 shown). Repeated 2 times. (G) Lungs excised at 10 days post-RO injection of mice described in (E), subjected to bioluminescent imaging for Firefly luciferase, repeated twice. (H) Quantification of metastatic burden in lungs of mice from (E). Total luciferase fluorescent flux quantified (photons [ps]/sec). *p < 0.05 by unpaired two-tailed t-test.

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Western Blot, Transformation Assay, Stable Transfection, Transfection, Staining, Two Tailed Test, Migration, Wound Healing Assay, Injection, Expressing, Luciferase, Imaging

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-human/mouse DAP5 antibody , BD Biosciences , Cat# 610291/RRID:AB_397685.

Techniques: Recombinant, Staining, Protease Inhibitor, Virus, SYBR Green Assay, Bicinchoninic Acid Protein Assay, MTT Assay, Software, Imaging

Journal: International journal of molecular sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

doi: 10.3390/ijms242417149

Figure Lengend Snippet: Figure 2. eIF4G2 participates in the translation of both POLGARF and POLG. (A) The contribution of eIF4G2 to translation was assessed using reporter mRNA transfection in 293T cells depleted of eIF4G2. Cells pretreated with either control or anti-eIF4G2 siRNAs for 72 h were transfected with in vitro transcribed m7G-capped and polyadenylated reporters. The Nluc-coding reference β-globin reporter mRNA was co-transfected with all of the reporters. The data are presented as ratios of normalized reporter expression in the eIF4G2-depleted to the control cells (the eIF4G2 KD effect). The knockdown effect < 1 corresponds to translation inhibition in the absence of eIF4G2. The translation of Stard7, Maf1, and UCP2 mRNAs has previously been shown to require eIF4G2. The reporter mRNAs with β-globin, SLC1A5, FN1, Moderna, and BioNTech 5′ UTR do not need eIF4G2 and serve as a negative control. For all eIF4G2 targets and β-globin reporters, the number of replicates exceeds 20. For the

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: Transfection, Control, In Vitro, Expressing, Knockdown, Inhibition, Negative Control

Journal: International journal of molecular sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

doi: 10.3390/ijms242417149

Figure Lengend Snippet: Figure 3. eIF4G2 promotes POLG and POLGARF translation. (A) In vitro transcribed m7G-capped and polyadenylated POLG reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted (siRNA#1) 293T cells along with the reference β-globin reporter mRNA (n ≥10). All assayed reporters were tested with either the wild-type or the disrupted stem-loop (referred to as SL intact or SL disrupted, respectively). The effect of the knockdown (eIF4G2 KD effect) is calculated by dividing the normalized reporter expression in eIF4G2-depleted cells by that in control cells. The knockdown effect < 1 reflects translation inhibition by the eIF4G2 depletion. The statistical significance is determined using the Mann–Whitney U test. Three and four asterisks stand for p < 0.001 and p < 0.0001, respectively. (B) Results of POLGARF reporter mRNA transfections (similar to panel A). (C) Schematic representation of the POLG reporters examined in panel A (not to scale). The POLG start codon (red bar) drives translation of the chimeric reporter protein, which consists of 15 N-terminal POLG amino acids (shown in pink) fused to the firefly luciferase (Fluc). The POLGARF CUG codon (the dark blue bar) drives the translation of an ORF that overlaps out of frame with the Fluc and encodes the 60-aa-long peptide with 33 N-terminal POLGARF amino acids (CUG-driven uORF is displayed in blue). The purple box depicts the regulatory uORF. The start codons are shown in corresponding color bars, and black bars depict the stop codons. The stem-loop is omitted for the sake of readability. Crosses display the positions of the upstream (with respect to the POLG ORF) start codons that were substituted for the stop codons in the corresponding reporters. In the reporter mRNA called “fused”, the uORF is fused in frame to the chimeric POLG/Fluc sequence. This resulting chimaera evaluates the role of eIF4G2 in the POLG/POLGARF mRNA translation initiation from the 5′-end to the uAUG. (D) Panel D is similar to panel C, with the exception that POLGARF reporter mRNAs are displayed. The POLGARF CUG start codon drives translation of a chimeric reporter protein, consisting of 33 N-terminal POLGARF amino acids (shown in blue) fused to the Fluc. The POLG start codon is out of frame with Fluc and provides translation of a 20-amino-acid-long stub (shown in pink).

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Expressing, Control, Inhibition, MANN-WHITNEY, Luciferase, Sequencing

Journal: International journal of molecular sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

doi: 10.3390/ijms242417149

Figure Lengend Snippet: Figure 5. eIF4G2 promotes both leaky scanning and reinitiation on POLG and POLGARF mRNAs. (A) In vitro transcribed m7G-capped and polyadenylated POLG reporters with the indicated wild- type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted 293T cells alongside reference β-globin reporter mRNA (n ≥10). The knockdown was accomplished using siRNA #1 against eIF4G2. Almost all assayed reporters were tested with either a wild-type or a disrupted stem- loop sequence (referred to as “SL intact” and “SL disrupted”, respectively). The knockdown effect (eIF4G2 KD effect) is calculated by dividing normalized reporter expression in eIF4G2-depleted cells by normalized expression in control cells. The knockdown effect < 1 signifies a translation inhibition in the absence of eIF4G2. The statistical significance is determined using the Mann–Whitney U test. The asterisks indicate p-value (one, two, three, and four asterisks stand for p < 0.05, p < 0.01, p < 0.001, and p < 0.001, respectively). (B) Results of POLGARF reporter mRNA transfections (similar to panel (A)). (C) Schematic representation of the POLG reporters examined in panel A (not to scale). Ri- bosomes can reach the main POLG start codon via leaky scanning through the uAUG or by reinitiation after completing the translation of the uORF. To estimate the eIF4G2 contribution to the leaky scanning, the uORF stop codon was mutated so that the extended uORF became fused to the POLGARF ORF and overlapped out of frame with the firefly luciferase ORF (referred to as “uORF ext”). To significantly reduce the leaky scanning through the uORF, two extra start AUG codons (referred to as “uAUG x2”) were inserted in frame and near the uAUG codon. On such a 5′ UTR, ribosomes mostly reach the main

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Sequencing, Expressing, Control, Inhibition, MANN-WHITNEY, Luciferase

Journal: International journal of molecular sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

doi: 10.3390/ijms242417149

Figure Lengend Snippet: Figure 7. eIF4G2 promotes both leaky scanning and reinitiation on Stard7, Maf1, and UCP2 mRNAs. (A) In vitro transcribed m7G-capped and polyadenylated Maf1, Stard7, and UCP2 reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted (siRNA#1) 293T cells alongside reference β-globin reporter mRNA (n ≥10). The effect of the knockdown (eIF4G2 KD effect) is calculated by dividing normalized reporter expression in eIF4G2- depleted cells by normalized expression in control cells. The knockdown effect <1 shows translation inhibition in the absence of eIF4G2. (B) Panel B is similar to panel A, except siRNA #2 was used for the eIF4G2 depletion in 293T cells (n ≥10). (C) Schematic representation of the reporters examined in panels A, B, and E (not in scale). Stard7, Maf1, and UCP2 5′UTRs contain uORFs (shown in blue box). Dark blue and red bars depict uAUG and main start codons, respectively, and black bars depict stop codons. All analyzed 5′ UTRs were mutated to address leaky scanning and reinitiation separately. Leaky scanning is addressed using an mRNA reporter with an extended uORF that

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Expressing, Control, Inhibition

Journal: International journal of molecular sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

doi: 10.3390/ijms242417149

Figure Lengend Snippet: Figure 8. In vitro translation with eIF1. Reporter mRNAs were translated in S20 cell extract from Expi293F cells in the presence of eIF1 or a storage buffer (n ≥5). The Fluc activity (or Nluc in the case of the eIF1 reporter) was measured. Translation inhibition by eIF1 is demonstrated relative to reporter activity in the presence of a storage buffer. The dotted line at 1 represents translation that is completely unresponsive to eIF1. (A) eIF1 inhibits translation of reporter POLG mRNAs. (B) The effect of eIF1 on the translation of POLGARF reporter mRNAs. (C) The effect of eIF1 on the translation of β-globin, eIF5, eIF1, and eIF4G2 reporter mRNAs. The reporter mRNA for eIF1 and eIF4G2 has wild-type GUG start codons.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Activity Assay, Inhibition

Journal: International journal of molecular sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

doi: 10.3390/ijms242417149

Figure Lengend Snippet: Figure 9. The proposed eIF4G2′s roles in cap-dependent translation of mRNA with uORFs. (A) The initial steps of translation initiation occur in accordance with the conventional Kozak’s mechanism. The small ribosome subunit is recruited to the 5′ end of mRNA via interaction between m7G-cap and eIF4F. Then eIF4G1 facilitates scanning from the very 5′ end. The 80S ribosome is depicted translating the uORF. uORF translation leads to the need for eIF4G2 that can mediate both leaky scanning and reinitiation on a single mRNA at the same time. (B) The role of eIF4G2 in leaky scanning has been linked to a partial loss of eIF4G1 caused by collisions of the scanning complex and the 80S ribosome within the uORF. Through interaction with eIF4E, the eIF4G1 gets back to the m7G-cap, and the role of helicase is then assumed by the eIF4G2–eIF4A complex, thereby mediating leaky scanning. (C) eIF4G2 can also facilitate reinitiation on the same mRNA. eIF4G2 assists the post-terminating 80S ribosome to resume scanning and thus contributes to reinitiation. The scheme from [5] was elaborated.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: eIF4G2 participates in the translation of both POLGARF and POLG. ( A ) The contribution of eIF4G2 to translation was assessed using reporter mRNA transfection in 293T cells depleted of eIF4G2. Cells pretreated with either control or anti-eIF4G2 siRNAs for 72 h were transfected with in vitro transcribed m 7 G-capped and polyadenylated reporters. The Nluc-coding reference β-globin reporter mRNA was co-transfected with all of the reporters. The data are presented as ratios of normalized reporter expression in the eIF4G2-depleted to the control cells (the eIF4G2 KD effect). The knockdown effect < 1 corresponds to translation inhibition in the absence of eIF4G2. The translation of Stard7, Maf1, and UCP2 mRNAs has previously been shown to require eIF4G2. The reporter mRNAs with β-globin, SLC1A5, FN1, Moderna, and BioNTech 5′ UTR do not need eIF4G2 and serve as a negative control. For all eIF4G2 targets and β-globin reporters, the number of replicates exceeds 20. For the non-targets n ≥ 5. ( B ) Western blot analysis of the eIF4G2 knockdown in 293T cells, GAPDH as a loading control.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: Transfection, Control, In Vitro, Expressing, Knockdown, Inhibition, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: POLG and POLGARF translation is cap-dependent and unresistant to eIF2α phosphorylation. ( A ) 293T cells were treated with either vehicle (DMSO) or 1 μM of PP242 to activate eIF4E-BPs. Then, the cells were transfected with the indicated mRNA reporters along with β-globin reference mRNA coding for Nluc, as an appropriate internal reference ( n ≥ 6). The data are presented as ratios of normalized reporter expression in cells treated with 1 μM of PP242 to that in vehicle-treated cells. PP242 treatment inhibits reference β-globin mRNA translation by ~3.2-fold (shown by the dashed line). The dotted line at 1 corresponds to observed translation inhibition identical to that of the reference β-globin reporter. Translation of control globin, TGFβ1, eIF4G2 mRNA is known to be cap dependent. PTV behavior represents a totally PP242-unresponsive translation. ( B ) Panel B is similar to panel A, except 293T cells were treated with either vehicle (PBS) or 1 μM of thapsigargin to induce ER stress and subsequent eIF2α phosphorylation. Thapsigargin’s effect is calculated by dividing normalized reporter expression in cells treated with 1 μM of thapsigargin to that in vehicle-treated cells. A dashed line represents a 3.9-fold drop in reference β-globin mRNA translation under thapsigargin treatment. A dotted line at 1 represents reporter behavior identical to reference β-globin. ATF4 behavior corresponds to a completely thapsigargin-unresponsive translation and is shown as a positive control.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Transfection, Expressing, Inhibition, Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: eIF4G2 promotes POLG and POLGARF translation. ( A ) In vitro transcribed m 7 G-capped and polyadenylated POLG reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted (siRNA#1) 293T cells along with the reference β-globin reporter mRNA ( n ≥ 10). All assayed reporters were tested with either the wild-type or the disrupted stem-loop (referred to as SL intact or SL disrupted, respectively). The effect of the knockdown (eIF4G2 KD effect) is calculated by dividing the normalized reporter expression in eIF4G2-depleted cells by that in control cells. The knockdown effect < 1 reflects translation inhibition by the eIF4G2 depletion. The statistical significance is determined using the Mann–Whitney U test. Three and four asterisks stand for p < 0.001 and p < 0.0001, respectively. ( B ) Results of POLGARF reporter mRNA transfections (similar to panel A). ( C ) Schematic representation of the POLG reporters examined in panel A (not to scale). The POLG start codon (red bar) drives translation of the chimeric reporter protein, which consists of 15 N-terminal POLG amino acids (shown in pink) fused to the firefly luciferase (Fluc). The POLGARF CUG codon (the dark blue bar) drives the translation of an ORF that overlaps out of frame with the Fluc and encodes the 60-aa-long peptide with 33 N-terminal POLGARF amino acids (CUG-driven uORF is displayed in blue). The purple box depicts the regulatory uORF. The start codons are shown in corresponding color bars, and black bars depict the stop codons. The stem-loop is omitted for the sake of readability. Crosses display the positions of the upstream (with respect to the POLG ORF) start codons that were substituted for the stop codons in the corresponding reporters. In the reporter mRNA called “fused”, the uORF is fused in frame to the chimeric POLG/Fluc sequence. This resulting chimaera evaluates the role of eIF4G2 in the POLG/POLGARF mRNA translation initiation from the 5′-end to the uAUG. ( D ) Panel D is similar to panel C, with the exception that POLGARF reporter mRNAs are displayed. The POLGARF CUG start codon drives translation of a chimeric reporter protein, consisting of 33 N-terminal POLGARF amino acids (shown in blue) fused to the Fluc. The POLG start codon is out of frame with Fluc and provides translation of a 20-amino-acid-long stub (shown in pink).

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Expressing, Control, Inhibition, MANN-WHITNEY, Luciferase, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: eIF4G2 promotes POLG and POLGARF translation. ( A ) In vitro transcribed m 7 G-capped and polyadenylated POLG reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted 293T cells alongside reference β-globin reporter mRNA ( n ≥ 8). The knockdown was accomplished using siRNA #2 against eIF4G2. All assayed reporters were tested with either a wild-type or a disrupted stem-loop sequence (referred to as SL intact and SL disrupted, respectively). The knockdown effect (eIF4G2 KD effect) is calculated by dividing normalized reporter expression in eIF4G2-depleted cells by normalized expression in control cells. The knockdown effect < 1 corresponds to translation inhibition in the absence of eIF4G2. The statistical significance is determined using the Mann–Whitney U test. Four asterisks correspond to p < 0.0001. ( B ) Results of reporter POLGARF mRNA transfections (similar to panel ( A )). Four and two asterisks correspond to p < 0.0001 and p < 0.01, respectively.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Sequencing, Expressing, Control, Inhibition, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: eIF4G2 promotes both leaky scanning and reinitiation on POLG and POLGARF mRNAs. ( A ) In vitro transcribed m 7 G-capped and polyadenylated POLG reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted 293T cells alongside reference β-globin reporter mRNA ( n ≥ 10). The knockdown was accomplished using siRNA #1 against eIF4G2. Almost all assayed reporters were tested with either a wild-type or a disrupted stem-loop sequence (referred to as “SL intact” and “SL disrupted”, respectively). The knockdown effect (eIF4G2 KD effect) is calculated by dividing normalized reporter expression in eIF4G2-depleted cells by normalized expression in control cells. The knockdown effect < 1 signifies a translation inhibition in the absence of eIF4G2. The statistical significance is determined using the Mann–Whitney U test. The asterisks indicate p -value (one, two, three, and four asterisks stand for p < 0.05, p < 0.01, p < 0.001, and p < 0.001, respectively). ( B ) Results of POLGARF reporter mRNA transfections (similar to panel A ). ( C ) Schematic representation of the POLG reporters examined in panel A (not to scale). Ribosomes can reach the main POLG start codon via leaky scanning through the uAUG or by reinitiation after completing the translation of the uORF. To estimate the eIF4G2 contribution to the leaky scanning, the uORF stop codon was mutated so that the extended uORF became fused to the POLGARF ORF and overlapped out of frame with the firefly luciferase ORF (referred to as “uORF ext”). To significantly reduce the leaky scanning through the uORF, two extra start AUG codons (referred to as “uAUG x2”) were inserted in frame and near the uAUG codon. On such a 5′ UTR, ribosomes mostly reach the main POLG start codon via reinitiation. Both mutations were introduced into the POLG 5′ UTR (referred to as “uAUG x2 & uORF ext”) to estimate the scanning complexes’ retention at the uORF in-frame start codons. The reinitiated scanning complexes on their way to the POLG start codon might interfere with the 80S ribosomes on the POLGARF ORF, thus increasing the need for eIF4G2. To estimate if this is the case, we compare the eIF4G2 contribution to reinitiation on the “POLG uAUG x2” reporter with that on the corresponding reporter mRNA with a mutated POLGARF start codon (referred to as “uAUG x2 & CUGm”). The start codons are shown in corresponding color bars, and black bars depict stop codons. ( D ) Panel D is similar to panel C, with the exception that POLGARF reporter mRNAs are displayed. The uORF stop codon was mutated so that the extended uORF became fused to the POLG ORF stub and overlapped out of frame with the firefly luciferase ORF (referred to as “uORF ext”). The POLGARF “uAUG x2” and “uAUG x2 & uORF ext” reporters are constructed similarly to corresponding POLG reporters. ( E ) Contribution of leaky scanning and reinitiation to translation of POLG and POLGARF mRNAs. Relative translation efficiency is calculated by dividing the normalized expression of a mutant reporter (designated as “uAUG x2” or “uORF ext” to address leaky scanning and reinitiation, respectively) by the normalized expression of a wild-type reporter. When reporters with disrupted stem-loops were used, the relative translation efficiency is shown with respect to corresponding reporters having wild-type start codons and a disrupted stem-loop. The dotted line at 1 corresponds to translation efficiency identical to that of the wild-type reporter mRNA. Note that the contribution of the reinitiation mode can be estimated through the subtraction of leaky scanning mode from 1. The “uAUG x2” reporter constructs arguably reflect the upper estimate for the contribution of reinitiation mode on the wild-type mRNA. The statistical significance was determined using the Mann–Whitney U test. The asterisks indicate p -value (four, three, and one asterisks stand for p < 0.0001, p < 0.001, and p < 0.01, respectively). ( F ) Reinitiating ribosomes mainly bypass the POLGARF CUG start codon. Relative translation efficiency is calculated in the same way as in Panel E, and statistical significance is determined similarly. Asterisks denote p < 0.0001.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Sequencing, Expressing, Control, Inhibition, MANN-WHITNEY, Luciferase, Construct, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: eIF4G2 promotes both leaky scanning and reinitiation on POLG and POLGARF mRNAs. ( A ) In vitro transcribed m 7 G-capped and polyadenylated POLG reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted 293T cells alongside reference β-globin reporter mRNA ( n ≥ 10). The knockdown was accomplished using siRNA #2 against eIF4G2. Almost all assayed reporters were tested with either a wild-type or a disrupted stem-loop sequence (referred to as SL intact and SL disrupted, respectively). The knockdown effect (eIF4G2 KD effect) is calculated by dividing normalized reporter expression in eIF4G2-depleted cells by normalized expression in control cells. The knockdown effect < 1 corresponds to translation inhibition in the absence of eIF4G2. The statistical significance is determined using the Mann–Whitney U test (one, two, three, and four asterisks stand for p < 0.05, p < 0.01, p < 0.001, and p < 0.001, respectively). ( B ) Results of reporter POLGARF mRNA transfections (similar to panel ( A )). Asterisks stand for p < 0.001.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Sequencing, Expressing, Control, Inhibition, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: eIF4G2 promotes both leaky scanning and reinitiation on Stard7, Maf1, and UCP2 mRNAs. ( A ) In vitro transcribed m 7 G-capped and polyadenylated Maf1, Stard7, and UCP2 reporters with the indicated wild-type (WT) or mutated 5′ UTRs were transfected into mock- and eIF4G2-depleted (siRNA#1) 293T cells alongside reference β-globin reporter mRNA ( n ≥ 10). The effect of the knockdown (eIF4G2 KD effect) is calculated by dividing normalized reporter expression in eIF4G2-depleted cells by normalized expression in control cells. The knockdown effect <1 shows translation inhibition in the absence of eIF4G2. ( B ) Panel B is similar to panel A, except siRNA #2 was used for the eIF4G2 depletion in 293T cells (n ≥ 10). ( C ) Schematic representation of the reporters examined in panels A, B, and E (not in scale). Stard7, Maf1, and UCP2 5′UTRs contain uORFs (shown in blue box). Dark blue and red bars depict uAUG and main start codons, respectively, and black bars depict stop codons. All analyzed 5′ UTRs were mutated to address leaky scanning and reinitiation separately. Leaky scanning is addressed using an mRNA reporter with an extended uORF that overlaps significantly with the Fluc coding sequence (referred to as “uORF ext”). The insertion of two additional uAUGs into uORF (designated as “uAUG x2”) makes the reinitiation nearly the only way for ribosomes to reach the main start codon. To estimate the scanning complexes retention at the uORF in-frame start codons, both mutations were introduced into these 5′ UTRs (referred to as “uAUG x2 & uORF ext”). ( D ) Ribosome Decision Graphs representing translation as multiple ribosome paths through the wild-type or mutated 5′ UTRs. Boxes demonstrate ORFs. Circles depict branching points where the ribosome makes a “decision” of whether to initiate or not. The path of leaky scanning complexes towards the downstream start codon is shown in dark grey. Light grey paths represent the post-terminating small ribosome subunit that resumes scanning and can initiate on the main start codon (reinitiation path). The dotted paths represent the significant decrease in the corresponding mode of initiation. ( E ) The contributions of leaky scanning and reinitiation to the translation of Stard7, Maf1, and UCP2 mRNAs. Relative translation efficiency is calculated by dividing the normalized expression of a mutant reporter (designated as “uAUG x2” or “uORF ext” to address leaky scanning and reinitiation, respectively) by the normalized expression of a wild-type reporter ( n ≥ 10). The dotted line at 1 corresponds to translation efficiency identical to that of the wild-type reporter mRNA. Note that the contribution of the reinitiation mode can be estimated through subtraction of the leaky scanning mode from 1. The “uAUG x2” reporter constructs reflect the upper estimate for the contribution of the reinitiation mode on the wild-type mRNA.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Transfection, Knockdown, Expressing, Control, Inhibition, Sequencing, Mutagenesis, Construct

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: In vitro translation with eIF1. Reporter mRNAs were translated in S20 cell extract from Expi293F cells in the presence of eIF1 or a storage buffer ( n ≥ 5). The Fluc activity (or Nluc in the case of the eIF1 reporter) was measured. Translation inhibition by eIF1 is demonstrated relative to reporter activity in the presence of a storage buffer. The dotted line at 1 represents translation that is completely unresponsive to eIF1. ( A ) eIF1 inhibits translation of reporter POLG mRNAs. ( B ) The effect of eIF1 on the translation of POLGARF reporter mRNAs. ( C ) The effect of eIF1 on the translation of β-globin, eIF5, eIF1, and eIF4G2 reporter mRNAs. The reporter mRNA for eIF1 and eIF4G2 has wild-type GUG start codons.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques: In Vitro, Activity Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA

doi: 10.3390/ijms242417149

Figure Lengend Snippet: The proposed eIF4G2′s roles in cap-dependent translation of mRNA with uORFs. ( A ) The initial steps of translation initiation occur in accordance with the conventional Kozak’s mechanism. The small ribosome subunit is recruited to the 5′ end of mRNA via interaction between m 7 G-cap and eIF4F. Then eIF4G1 facilitates scanning from the very 5′ end. The 80S ribosome is depicted translating the uORF. uORF translation leads to the need for eIF4G2 that can mediate both leaky scanning and reinitiation on a single mRNA at the same time. ( B ) The role of eIF4G2 in leaky scanning has been linked to a partial loss of eIF4G1 caused by collisions of the scanning complex and the 80S ribosome within the uORF. Through interaction with eIF4E, the eIF4G1 gets back to the m 7 G-cap, and the role of helicase is then assumed by the eIF4G2–eIF4A complex, thereby mediating leaky scanning. ( C ) eIF4G2 can also facilitate reinitiation on the same mRNA. eIF4G2 assists the post-terminating 80S ribosome to resume scanning and thus contributes to reinitiation. The scheme from was elaborated.

Article Snippet: Antibodies against eIF4G2 (A302-249A), GAPDH (A300-639A), and eIF3d (A301-759A) were purchased from Bethyl laboratories (Montgomery, TX, USA).

Techniques:

Journal: Neurobiology of disease

Article Title: Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

doi: 10.1016/j.nbd.2023.106212

Figure Lengend Snippet: Fig. 1. Genetic ablation of DAP5 suppresses CGG repeat expansion-associated toxicity in a Drosophila model of FXTAS. (A) Domain architecture of eIF4Gs with their nomenclatures in human and Drosophila. Color-coded domains highlight binding/interacting regions for respective factors. (B) A schematic of Drosophila screening to evaluate the role of eIF4Gs in modulating rough eye phenotype caused by CGG repeat expansion. (C) Quantitation of uas-(CGG)90-EGFP eye phenotype with eIF4G modifiers. siNTC = non-targeting siRNA against luciferase (LUC) gene. Different siRNA/insertion lines for the same target are numbered (#1, #2 and #3). Violin plots show all rough eye scores for n ≥30 flies/genotype with solid lines representing the median value. One-way ANOVA with Dunnett’s multiple comparison test, **** P < 0.0001. (D) Representative pictures of fly eyes expressing uas-(CGG)90-EGFP under a GMR-GAL4 driver, with disruptions of various eIF4Gs.

Article Snippet: The following antibodies were used for western blots as specified for dilution in 5% non-fat milk; FLAG-M2 at 1:1000 dilution (mouse, Sigma F1804), 1:2500 β-Actin (mouse, Sigma A1978), 1:2500 GAPDH 6C5 (mouse, Santa Cruz sc32233), 1:1000 DAP5 (rabbit, CST 5169S), 1:1000 EIF3G (rabbit, Bethyl Laboratories, Inc. A301-757A-T), 1:1000 EIF3F (rabbit, Bethyl Laboratories, Inc. A303-005A-T), 1:1000 EIF2β (rabbit, Protein Tech 10,227–1-AP), 1:1000 GFP (mouse, Roche/Sigma 11,814,460,001).

Techniques: Binding Assay, Quantitation Assay, Luciferase, Comparison, Expressing

Journal: Neurobiology of disease

Article Title: Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

doi: 10.1016/j.nbd.2023.106212

Figure Lengend Snippet: Fig. 4. DAP5 knockdown reduces RAN translation product accumulation in Drosophila. (A) Representative photographs of Drosophila external eye showing accu mulation of GFP aggregates caused by (CGG)90-EGFP transgene expression (left). Converted images used to quantify total intensity of GFP puncta (right). (B) Western blots of the FMRpolyG-EGFP RAN translation product in ELAV-GS driven (CGG)90-EGFP transgene expressing flies with and without DAP5 knockdown. Total protein from three (n = 3 biological repeats) independent fly crosses run in three replicates per gel (technical replicates). All data points presented in the graphs. (C) Survival data presented as violin plots showing the distributions of day of death events for each modifier (n = 120–140/genotype) after activation post-eclosion. Statistical analysis: (A) One-way ANOVA with Dunnett’s multiple comparison test; (B, C) Two-tailed Student’s t-test with Welch’s correction, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The following antibodies were used for western blots as specified for dilution in 5% non-fat milk; FLAG-M2 at 1:1000 dilution (mouse, Sigma F1804), 1:2500 β-Actin (mouse, Sigma A1978), 1:2500 GAPDH 6C5 (mouse, Santa Cruz sc32233), 1:1000 DAP5 (rabbit, CST 5169S), 1:1000 EIF3G (rabbit, Bethyl Laboratories, Inc. A301-757A-T), 1:1000 EIF3F (rabbit, Bethyl Laboratories, Inc. A303-005A-T), 1:1000 EIF2β (rabbit, Protein Tech 10,227–1-AP), 1:1000 GFP (mouse, Roche/Sigma 11,814,460,001).

Techniques: Knockdown, Expressing, Western Blot, Activation Assay, Comparison, Two Tailed Test

Journal: Neurobiology of disease

Article Title: Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

doi: 10.1016/j.nbd.2023.106212

Figure Lengend Snippet: Fig. 6. Modulating DAP5 expression impacts CGG RAN translation in a cell-type specific manner. (A) Representative Western blot showing expression of a FLAG epitope-tagged exogenous DAP5 plasmid (DAP5-FLAG) in HEK293T cells. An exogenous EGFP expressing plasmid used for comparison with DAP5 overexpression. β-Actin is used as a loading control. (B) Relative expressions of CGG-nLUC following overexpression of DAP5-FLAG (n = 9/condition) compared to EGFP expression in HEK293T cells. (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in U2OS cells (n = 9/condition) following knockdown of DAP5. (D) Anti-FLAG immunoblots showing effects of DAP5 knockdown on expression of +1CGG (100)-nLuc-3xFLAG RAN translation reporter in U2OS cells. β-Actin is used as a loading control (n = 6/conditions). (E) Relative expression of AUG-nLuc and CGG-nLuc reporters in cultured RAT neurons (n = 9/condition) following knockdown of DAP5.

Article Snippet: The following antibodies were used for western blots as specified for dilution in 5% non-fat milk; FLAG-M2 at 1:1000 dilution (mouse, Sigma F1804), 1:2500 β-Actin (mouse, Sigma A1978), 1:2500 GAPDH 6C5 (mouse, Santa Cruz sc32233), 1:1000 DAP5 (rabbit, CST 5169S), 1:1000 EIF3G (rabbit, Bethyl Laboratories, Inc. A301-757A-T), 1:1000 EIF3F (rabbit, Bethyl Laboratories, Inc. A303-005A-T), 1:1000 EIF2β (rabbit, Protein Tech 10,227–1-AP), 1:1000 GFP (mouse, Roche/Sigma 11,814,460,001).

Techniques: Expressing, Western Blot, FLAG-tag, Plasmid Preparation, Comparison, Over Expression, Control, Knockdown, Cell Culture