Anti-eIF4A1 antibody

• WB

Project

Western blot - Anti-eIF4A1 antibody (AB31217)

All lanes:

Western blot - Anti-eIF4A1 antibody (ab31217) at 1 µg/mL

Lane 1:

Western blot - NIH/3T3 whole cell lysate (<a href='/en-us/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 10 µg

Lane 2:

MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 3:

Liver (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 4:

Western blot - Mouse pancreas tissue lysate - total protein (<a href='/en-us/products/tissue-lysates/mouse-pancreas-tissue-lysate-total-protein-ab29363'>ab29363</a>) at 10 µg

Lane 5:

Testis (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 6:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 46 kDa

Observed band size: 47 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-eIF4A1 antibody (AB31217)

ICC/IF image of ab31217 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab31217, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used to quench autofluorescence. 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

• IP

Unknown

Immunoprecipitation - Anti-eIF4A1 antibody (AB31217)

eIF4A1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to eIF4A1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31217.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 47kDa : eIF4A1

All lanes:

Immunoprecipitation - Anti-eIF4A1 antibody (ab31217)

Predicted band size: 46 kDa

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• WB

Project

Western blot - Anti-eIF4A1 antibody (AB31217)

All lanes:

Western blot - Anti-eIF4A1 antibody (ab31217) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

Western blot - Jurkat whole cell lysate (<a href='/en-us/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 20 µg

Lane 3:

Western blot - A-431 whole cell lysate (<a href='/en-us/products/cell-lysates/a-431-whole-cell-lysate-ab7909'>ab7909</a>) at 20 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Predicted band size: 46 kDa

Observed band size: 47 kDa

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• WB

CiteAb

Western blot - Anti-eIF4A1 antibody (AB31217)

Western Blotting using Anti-eIF4A1 antibody, ab31217. Publication image from Zanivan, S. et al., 2019, Genome Biol, 31791371. Legend direct from paper.

eIF4A2 represses translation at initiation. a Western blot demonstrates specificity of immunoprecipitation for each protein from a representative experiment. Input represents 10% of lysate used in IP. Asterisk denotes non-specific signal from IgG. Venn diagram showing numbers of mRNAs significantly (FDR < 0.05) enriched over input in the respective endogenous RIP-Seq (n = 3). b Differential association with polysomes of mRNAs bound to one of the two proteins or both compared to all mRNAs identified in the RIP-Seq experiment. Relative distribution of mRNAs on sucrose density gradients was calculated from RNA-Seq analysis of the subpolysomal and polysomal fractions in a separate experiment (n = 4) by subtracting counts per million between the two fractions. Significance calculated using Dunn’s test with Bonferroni’s correction. c Differential ribosome occupancy of eIF4A2- and eIF4A1-bound messages. Ribosome profiling was performed in HEK293 lysates (n = 3). Ribosome occupancy for each mRNA at each nt position is calculated as the number of ribosome footprints normalized to the mRNA abundance (transcripts per million—TPM). Shown is the mean number of normalized ribosome footprints 75 codons downstream of the AUG and upstream of the STOP codon. d iBAQ—intensity-based absolute quantification [48]—of protein abundance in control conditions in pulsed SILAC for bound mRNAs. e Proportions of mRNAs bound by eIF4A1 and eIF4A2 predicted to be miRNA targets by the Targetscan algorithm. f eIF4A2-bound mRNAs have increased ribosome occupancy in the last 50 nt, but not in the first 50 nt of the 5′UTR. The RPF coverage was normalized for the abundance of the mRNAs (TPM). g Translation of the main AUG start codon is repressed by activation of uORFs in eIF4A2-bound mRNAs. Global translation initiation sequencing (GTI-seq) data from Lee et al. [49], also conducted in HEK293 cells, was used to assess the translation from uORFs in the groups of mRNAs bound by either eIF4A1, eIF4A2, or both. The stacked bars represent the proportions of the groups of mRNAs with active translation from the annotated translation start site, upstream start sites, or both

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• WB

CiteAb

Western blot - Anti-eIF4A1 antibody (AB31217)

Western Blotting using Anti-eIF4A1 antibody, ab31217. Publication image from Tebaldi, T. et al., 2018, Mol Cell, 30029004. Legend direct from paper.

HuD Enhancement of Global and Target-Specific Translation Efficiency Does Not Depend on the mTORC1 Pathway(A) Left : western blot analysis of Rps6 and Eif4ebp1 phosphorylation following serum deprivation (8 hr) in NSC-34 cells.(B) Measurement of global TE by sucrose gradient centrifugation in the following conditions : control, starvation, and starvation coupled with HuD overexpression.(C) TE quantification of selected mTOR-responsive mRNAs in control, starvation, and starvation coupled with HuD overexpression conditions. Target-specific TE is the ratio between polysomal and total RNA changes measured by RT-qPCR. Gapdh and Als2 were used as reference genes.(D) Western blot analysis of Eef1a1 and Eif4a3 in NSC-34 cells collected in three different conditions : control, starvation, and starvation with HuD overexpression.(E) Barplot displaying normalized luciferase intensity values in HEK293 cells transiently transfected with HuD, relative to transient transfection of the empty vector. Cells were co-transfected with wild-type (WT) or mutated (MUT) TOP motif bearing luciferase vectors with the 3′UTR of Eef1a1 (HuD target) or Eif4a3 (negative control).In (A)–(E), data are represented as mean ± SEM t test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. In (A)–(C), “Starvation” was compared to “Control,” and “Starvation + HuD overexpression” was compared to “Starvation” for testing statistical significance.See also Figure S3.

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