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Santa Cruz Biotechnology
eif4a3  Eif4a3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more https://www.bioz.com/result/eif4a3/product/Santa Cruz Biotechnology Average 93 stars, based on 1 article reviews
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Proteintech
eif4a3 depletion  Eif4a3 Depletion, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more https://www.bioz.com/result/eif4a3 depletion/product/Proteintech Average 95 stars, based on 1 article reviews
eif4a3 depletion - by Bioz Stars,
2026-02
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Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 1. eIF4A3 interacts with eIF3g. ( A ) The yeast two-hybrid assay using BD-eIF4A3 and the indicated AD-fused preys. Transformed yeast cells were spread on a selective medium lacking leucine and tryptophan (SD-LW) to select cotransformants. In our system, specific interactions between BD-eIF4A3 and the AD-library were expected to yield a visible blue color (LacZ expression in the filter assay), and the growth on a medium lacking uracil (SD-LWU) and on a medium lacking adenine (SD-LWA). A known interaction, homodimerization of polypyrimidine tract-binding protein, served as the positive control (+). The empty vectors expressing BD or AD served as the negative control ( −). ( B ) The yeast two-hybrid assay involving BD-eIF4A3 and AD-eIF3 subunits (all human proteins). ( C ) F ar-w estern blotting analysis. Purified rabbit and human eIF3 complexes and bovine serum albumin (BSA, which served as a negative control) were resolved by SDS–PAGE. Next, the purified recombinant eIF4A3 and the α-His antibody were used as a probe and primary antibody, respectively. The representative images of Coomassie blue staining and a far-western blot are shown in the left and right panels, respectiv ely. B ecause eIF3g and eIF3h comigrated in the SDS–PAGE gel under our conditions, w e could not distinguish a specific interactor of eIF4A3. FW, f ar-w estern blotting; WB, w estern blotting. ( D ) T he PLA assa y to e v aluate the interaction betw een EJC component and eIF3 components. HeLa cells either depleted or not depleted of the indicated protein were subjected to the PLA assay. The antibodies raised in a mouse or rabbit are colored y ello w or red, respectively. mIgG, nonspecific mouse IgG; rIgG, nonspecific rabbit IgG; scale bar, 10 μm. ( E ) Mapping of the domains of eIF3g required for its interaction with eIF4A3. The results obtained from IP experiments with FLAG-eIF4A3 and either GST-eIF3g or its deletion variant (Supplementary Figure S2) are summarized. ( F ) The GST pull-down assay. HEK293T cells either undepleted or depleted of eIF3g were transiently transfected with a plasmid expressing either GST or GST-eIF4A3. Representative images from at least two biological replicates ( n = 2) are presented in each panel.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques: Y2H Assay, Transformation Assay, Expressing, Binding Assay, Positive Control, Negative Control, Purification, SDS Page, Recombinant, Staining, Far Western Blot, Variant Assay, Pull Down Assay, Transfection, Plasmid Preparation
Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 2. An eIF4A3 tethered to the 5 ′ UTR drives the internal initiation of translation. ( A ) Schematic representation of tethering reporter mRNAs using 12 repeats of B o xB and λN system. AUG, a translation initiation codon; UAA, a translation stop codon; SL, a strong stem-loop structure; RLuc, Renilla luciferase; FLuc, firefly luciferase. ( B ) Artificial tethering of a core EJC component or eIF3g. HEK293T cells depleted of either eIF4A3 or eIF3g were cotransfected with a tethering reporter plasmid expressing SL-BoxB-R mRNA, an indicated effector plasmid and a reference plasmid (pGL3-control) expressing FLuc mRNA. Two days after transfection, cell extracts were subjected to dual luciferase assays. RLuc activities were normalized to FLuc activities. The normalized levels of RLuc activity in the presence of λN-HA-GFP were arbitrarily set to 1.0. Statistical significance between tethered λN-HA-GFP and other tethered proteins was calculated in each condition, and the corresponding P values are indicated by red asterisks. In addition, statistical significance among the depleted conditions was calculated, and the P values are indicated by blue asterisks with lines; n = 4; ** P < 0.01; #, not significant. ( C ) The effect of a tethered eIF4A3 on RLuc expression in cells depleted of one of EJC core components. As performed in ( B ), except that cells depleted of the indicated protein were transiently cotransfected with a plasmid expressing SL-BoxB-R mRNA, an indicated effector plasmid and pGL3-control; n = 3; ** P < 0.01. ( D ) The effect of tethered eIF4A3 on SL-BoxB-R mRNA in cells depleted of one of the eIF3 components; n = 3; * P < 0.05. ( E ) The impact of the tethering of the eIF4A3-WT or its variant. HEK293T cells were transiently expressed with a plasmid expressing SL-B o xB-R mRNA, an effector plasmid expressing one of the eIF4A3 variants and pGL3-control. Statistical significance between tethered λN-HA-GFP and other tethered proteins was calculated, and the corresponding P values are indicated by red asterisks. In addition, statistical significance between tethered λN-HA-eIF4A3-WT and its variant was calculated, and the P values are indicated by blue asterisks with lines; n = 3; ** P < 0.01; * P < 0.01; #, not significant. ( F ) Specific interaction of eIF4A3 variants with other EJC core components and eIF3g. Total-cell extracts of HEK293T cells transiently expressing FLAG-eIF4A3-WT or its variant were subjected to IP experiments using α-FLAG antibody. Co-immunopurified proteins were analyzed by western blotting. Representative images ( n = 2) are presented in the panel.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques: Luciferase, Plasmid Preparation, Expressing, Control, Transfection, Activity Assay, Variant Assay, Western Blot
Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 4. An eIF4A3 drives internal translation in the in vitro system. ( A ) The effect of an SL on in vitro translation. In vitro -synthesized BoxB-R or SL-B o xB-R RNA (either capped or uncapped) and in vitro -synthesized capped FLuc RNA were mixed with the cytoplasmic extracts of HeLa cells. RLuc activities were normalized to FLuc activities. The normalized levels of RLuc activity obtained from B o xB-R RNA were arbitrarily set to 1; n = 3; ** P < 0.01. ( B ) The in vitro translation assay using in vitro -synthesized linear reporter RNAs. In vitro -synthesized reporter RNA (SL-BoxB-R mRNA, either capped or uncapped) and in vitro -synthesized capped FLuc RNA were mixed with the cytoplasmic extracts of HeLa cells expressing the indicated effector protein. RLuc activity was normalized to FLuc activity. The normalized levels of RLuc activity in the extracts of the cells expressing λN-HA-GFP were arbitrarily set to 1; n = 3; * P < 0.05; ** P < 0.01. ( C ) A schematic diagram of in vitro transcription f ollo w ed b y in vitro circularization. ( D ) Validation of in vitro -synthesized circRNAs by denaturing agarose gel electrophoresis. To maximize the yield of circRNAs, we carried out the circularization reaction t wice. The det ails are described in the Methods section. ( E ) The in vitro translation assay using in vitro -synthesized circRNA s. A s performed in panels ( A ) and ( B ), e x cept that in vitro -synthesized circRNAs were mixed with the cytoplasmic extracts of HeLa cells expressing the indicated effector protein; n = 3; ** P < 0.01. ( F ) The effect of the tethered EJC on the translation of in vitro -synthesized circRNAs. As performed in ( E ), except that in vitro -synthesiz ed Circ-B o xB-R RNAs w ere mix ed with the cytoplasmic e xtracts of HeLa cells e xpressing the indicated effector protein; n = 6; ** P < 0.01.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques: In Vitro, Synthesized, Activity Assay, Expressing, Biomarker Discovery, Agarose Gel Electrophoresis
Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 5. Polysomal association of endogenous circRNAs depends on the eIF4A3 and is resistant to serum depletion. (A, B) The effect of Puro treatment on relative polysomal distributions of endogenous circRNAs. Subpolysomal fractions (containing subunits the 40S, 60S and 80S) and polysome fractions of HeLa cells either treated or not treated with Puro for 2 h were pooled. Next, in vitro -synthesized FLuc RNA, as a spike-in control, was added to the pooled fractions. ( A ) Polysome profiles of cytoplasmic extracts of HeLa cells either treated or not treated with Puro. ( B ) Quantitative representation of endogenous circRNAs in subpolysomal and polysomal fractions. The endogenous circRNAs in the subpolysomal and polysomal fractions were quantitated by RT-qPCRs using circRNA-specific divergent oligonucleotides; n = 3; ** P < 0.01; * P < 0.05; #, not significant. ( C–E ) The change in relative polysomal distributions of endogenous circRNAs after downregulation of eIF4A3 and complementation with siRNA-resistant FLAG-eIF4A3. ( C ) Western blotting validating specific downregulation of endogenous eIF4A3 and induction of FLAG-eIF4A3 up to a level comparable to that of endogenous eIF4A3. ( D ) Polysome profiles (upper) and relative distributions of endogenous proteins (lower). ( E ) Relative polysomal distributions of endogenous circRNAs. After polysome fractionation, in vitro -synthesized FLuc RNA, as a spike-in control, was added to the subpolysomal and polysomal fractions. The endogenous circRNAs in the subpolysomal and polysomal fractions were quantitated by RT-qPCRs using divergent oligonucleotides. Next, the levels were normalized to those of FLuc RNA. Relative distributions of endogenous circRNAs in the subpolysomal and polysomal fractions are presented as percentages; n = 3; * P < 0.05; ** P < 0.01; #, not significant.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques: Serum Depletion, In Vitro, Synthesized, Control, Western Blot, Fractionation
Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 6. Internal initiation of translation driven by eIF4A3 is resistant to cellular stresses. ( A–C ) Lack of significant change in relative polysomal distributions of endogenous circRNAs after serum depletion. As performed in Figure 5 A, B, except that HeLa cells were incubated in either a serum-containing or serum-free medium for 24 h before cell harvesting. ( A ) Western blotting showing 4E-BP1 dephosphorylation induced by serum depletion. ( B ) Polysome profiles (upper) and the relative distributions of endogenous proteins (lo w er). ( C ) R elativ e poly somal distributions of endogenous circRNAs; n = 3; ** P < 0.01; #, not significant. (D, E) The relative resistance of eIF4A3-mediated internal translation of linear mRNAs in response to serum depletion. HEK293T cells transiently e xpressing SL-B o xB-R mRNA, FLuc mRNA and either λN-HA-GFP or λN-HA-eIF4A3 were incubated in either a serum-containing or serum-free medium for 24 h before cell harvesting. ( D ) Western blots showing inefficient phosphorylation of 4E-BP1 on serum depletion. ( E ) Dual luciferase assay. RLuc and FLuc activities w ere normaliz ed to total protein amounts. T he normaliz ed le v els of RLuc and FLuc activities obtained from the cells expressing λN-HA-GFP in the serum-containing media were arbitrarily set to 1.0; n = 3; ** P < 0.01; #, not significant. (F, G) The relative resistance of eIF4A3-mediated internal translation of linear mRNAs in response to 4E-BP1 overexpression. HeLa cells were transiently expressed with SL-BoxB-R mRNA, FLuc mRNA, either λN-HA-GFP or λN-HA-eIF4A3, and either MYC or MYC-4E-BP1. ( F ) Western blots showing o v ere xpressed MYC-4E-BP1. ( G ) Dual luciferase assay. RLuc and FLuc activities w ere normaliz ed to total protein amounts. T he normaliz ed le v els of RLuc and FLuc activities obtained from the cells expressing λN-HA-GFP and MYC were arbitrarily set to 1.0; n = 3; ** P < 0.01; #, not significant.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques: Serum Depletion, Incubation, Cell Harvesting, Western Blot, De-Phosphorylation Assay, Phospho-proteomics, Luciferase, Expressing, Over Expression
Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 7. Polysomal enrichment of endogenous circRNAs is dependent on eIF4A3 at the transcriptome level. ( A ) The experimental scheme of polysome fractionation procedures f ollo w ed b y circRNA microarra y. Cytoplasmic e xtracts of HeLa cells, either depleted or not depleted of eIF4A3, w ere subjected to polysome fractionation. Next, RNA samples purified from either the pooled subpolysomal or polysomal fractions were subjected to the circRNA microarra y analy sis; n = 2. ( B ) Scatter plots of the relativ e change in poly somal association of circRNAs after eIF4A3 do wnregulation. T he x - and y -ax es represent the relative percentage distributions of circRNAs in the polysomal fraction in the undepleted cells and eIF4A3-depleted cells, respectiv ely. T he dots corresponding to cGSE1, cCTNNB1 and cZNF609 are depicted in cyan. Three types of cCTNNB1 were observed in our analysis. Statistical analysis w as perf ormed using the Wilco x on signed-rank test. ( C ) Scatter plots sho wing a correlation betw een the poly somal association of circRNAs and eIF4A3 dependency. The x -axis represents the relative percentage distributions of circRNAs in the polysomal fraction in the undepleted cells. The y -axis denotes the relative ratio (on the log 2 scale) of polysomal to subpolysomal level of circRNAs (PS ratio) in eIF4A3-depleted cells to PS ratio in the undepleted cells. (D, E) Scatter plots showing the relative change in polysomal association of circRNAs ( D ) or the effect of eIF4A3 downregulation on the PS ratios of circRNAs ( E ) according to its length. (F, G) Violin plots for the relative difference in the polysomal association of circRNAs ( F ) or effect of eIF4A3 downregulation on the PS ratios of circRNAs ( G ) according to their types. Statistical analysis was performed using one-way ANO V A with post hoc assessment via Tuk e y’s honestly significant difference test. ** P < 0.01, *** P < 0.001. (H, I) Violin plots for the relative difference in the polysomal association of circRNAs ( H ) or an effect of eIF4A3 downregulation on the PS ratios of circRNAs ( I ) according to the number of EJCs in circRNAs. *** P < 0.001.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques: Fractionation, Purification
Journal: Nucleic acids research
Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.
doi: 10.1093/nar/gkad763
Figure Lengend Snippet: Figure 9. A simplified model illustrating the eIF4A3-driven internal initiation of translation. In this study, we demonstrated that eIF4A3 alone possesses an intrinsic capability to initiate translation internally in an eIF3-dependent manner. Of note, this mode of translation is facilitated by other EJC core components. The relative translation efficiencies of 80S ribosome are indicated by the thickness of red arrows. ( A ) eIF4A3-driven internal initiation of translation in circRNAs. The 40S ribosome associates with circRNA with help of the eIF4A3 (or EJC)–eIF3 interaction and scans for AUG where it forms the 80S ribosome to translate the ORF in circRNA. On termination, the 40S either remains bound to circRNA, displaces the EJC and reinitiates (perhaps repeatedly) translation of the ORF in circRNA or undergoes full ribosome recycling to allow the eIF4A3 (or EJC)–eIF3 complex to mediate repeated internal initiation e v ents. ( B ) eIF4A3-driven internal initiation of translation in linear mRNA. Scanning or elongating ribosomes may displace all EJCs from mRNA s. O wing to this phenomenon, internal initiation driv en b y eIF4A3 w ould be suppressed under normal conditions. Ho w e v er, under stressful conditions where the general cap-dependent translation is compromised, the displacement would be inefficient and eIF4A3-mediated internal initiation of translation would become predominant, leading to protein synthesis from a downstream internal AUG (iAUG). See the Discussion section for more details.
Article Snippet: Primary antibodies against the following proteins were used for western blotting or IP: GST [catalog no. ( # ) A190-122A, Bethyl Laboratories], His tag ( 27–4710-01, GE Healthcare ) , HA tag ( 3F10; 11867431001, Roche ) , MYC tag ( 9E10; OP10L, Millipore ) ,
Techniques:
Journal: Genes & Development
Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons
doi: 10.1101/gad.353081.125
Figure Lengend Snippet: EIF4A3 depletion causes widespread skipping of neighboring exons. ( A ) Western blot showing EIF4A3 and GAPDH levels in SMASh-EIF4A3 HEK293T cells treated with DMSO or danoprevir for 24 h. ( B ) Number of altered splicing events by type after EIF4A3 depletion. ( C ) ΔPSI ( X -axis) versus event probability ( Y -axis; Whippet score) for cassette exons; color reflects local point density (2D Gaussian KDE). Threshold: probability ≥0.9 and ΔPSI ≥10% (mean of three biological replicates). ( D ) Histogram of differentially spliced exons per gene. ( E ) ΔPSI correlation of significantly altered exons from the same gene; the heat map shows point density. ( F ) Same as E but with randomly paired exons. ( G ) Bar chart showing relative positions of similarly affected exons (ΔPSI difference ≤0.02). (Yellow) adjacent, (orange) distal.
Article Snippet: To test
Techniques: Western Blot
Journal: Genes & Development
Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons
doi: 10.1101/gad.353081.125
Figure Lengend Snippet: Coordinated inclusion of neighboring exons requires multiple EJC components. ( A ) Western blot showing EIF4A3 overexpression relative to GAPDH in HEK293 Flp-In T-REx cells after induction with doxycycline for 48 h. ( B ) ΔPSI ( X -axis) versus event probability ( Y -axis; Whippet score) for cassette exons upon EIF4A3 overexpression; color reflects local point density (2D Gaussian KDE). Threshold: probability ≥0.9 and ΔPSI ≥10% (mean of three replicates). ( C ) Histogram of differentially spliced exons per gene in siRNA knockdowns of EJC components in HeLa cells. ( D ) ΔPSI correlation of significantly altered exons from the same gene; the heat map shows point density.
Article Snippet: To test
Techniques: Western Blot, Over Expression
Journal: Genes & Development
Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons
doi: 10.1101/gad.353081.125
Figure Lengend Snippet: The EJC is required for inclusion of exon blocks at the single transcript level. ( A ) Genome browser shot of individual long reads of mRNAs mapped to the DDX56 gene, showing control ( left ) and EIF4A3 depletion ( right ). Reference (ref.) gene is at the top , with coordinated block exons in orange. Reads are proportionally downsampled to 50 reads (total number of reads is indicated below the reads), with reads where exon blocks are skipped in orange and all other reads in green. The percentage of block-skipping reads is indicated at the right of reads. ( B ) Like A but for the FUS gene. ( C ) Schematic of block-skipping junctions, with the coordinated exon block in yellow (internal introns in orange) and surrounding exons in light blue. ( D ) Violin box plot illustrating the distribution of reads per million (RPM) spanning block-skipping junctions in control and EIF4A3 depletion conditions in mRNA long-read sequencing data sets (mean of three biological replicates). Values >60 were capped to avoid extreme outliers distorting the visualization.
Article Snippet: To test
Techniques: Control, Blocking Assay, Sequencing
Journal: Genes & Development
Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons
doi: 10.1101/gad.353081.125
Figure Lengend Snippet: EJC-dependent block exon inclusion occurs cotranscriptionally. ( A ) Illustration of the cell fractionation steps yielding nascent RNA (nRNA). ( B ) Western blot showing a successful cell fractionation, with tubulin as a cytoplasmic marker and RNA polymerase II (Pol 2) as a chromatin marker. ( C ) Schematic of block-skipping junctions. ( D ) Sashimi plot of nRNA short reads (green) aligned to the ACACA gene. The block exon region in yellow–orange is indicated in the reference gene below . Block-skipping junction read coverage is highlighted in yellow. ( E ) Like D but for the DDX56 gene. ( F ) Violin plot showing normalized read count in reads per million (RPM) in nRNA short reads spanning block-skipping junctions in control and EIF4A3 depletion conditions for three biological replicates (rep). ( G ) Scatter plot showing correlation of RPM for block exon-skipping junctions in mRNA versus nRNA short-read sequencing data sets. Pearson correlation and P -value are indicated in the top left of the plot.
Article Snippet: To test
Techniques: Blocking Assay, Cell Fractionation, Western Blot, Marker, Control, Sequencing
Journal: Genes & Development
Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons
doi: 10.1101/gad.353081.125
Figure Lengend Snippet: Introns within exon blocks are spliced out before the whole block is spliced in. ( A ) ΔSPI ( X -axis) versus P -value ( Y -axis); color reflects local point density (2D Gaussian KDE). Threshold: P -value ≤0.01 and ΔSPI ≥10% (mean of three replicates). ( B ) Normalized frequency histogram of SPI values for block internal introns (orange), block-flanking introns (dark green), and other introns (gray) in control ( left ) and EIF4A3 depletion ( right ) conditions. ( C ) Splicing order of introns surrounding the first (orange) and last (green) exons of coordinated exon blocks in control cells. The Y -axis is probability density (kernel density estimate), and the X -axis is fraction of upstream intron spliced first ( F UPFI ).
Article Snippet: To test
Techniques: Blocking Assay, Control