Anti-Argonaute-2抗体[2E12-1C9] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Ago2 / eIF2C2 antibody (ab57113) used in immunofluorescence at 10ug/ml on HeLa cells.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Ago2 / eIF2C2 antibody (ab57113) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human stomach.

• Flow Cyt

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Flow Cytometry - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Overlay histogram showing HeLa cells stained with ab57113 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab57113, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

This image was generated using the ascites version of the product.

• WB

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Western blot - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Western Blot detection against the recombinant fragment imunogen (68 KDa for a.a. ~377 plus GST tag +26 kDa).

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (ab57113)

Predicted band size: 97 kDa

Observed band size: 68 kDa

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• WB

CiteAb

Western blot - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Western Blotting using Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free, ab57113. Publication image from Gygi, S. P. et al., 2015, Nat Commun, 26018492. Legend direct from paper.

eIF1A interacts with Ago2(a) Proteins co-immunoprecipitated with Flag-HA-Ago2 from HEK293 cells were separated by SDS-PAGE (silver staining) and analyzed by mass spectrometry. eIF1A is a novel Ago2 interacting protein (* : detected peptides of eIF1A). (b) Fluorescence immunostaining was performed with anti-eIF1A (monoclonal) and anti-Ago2 (monoclonal) antibodies and DAPI (4′,6-diamidino-2-phenylindole) in HEK293 and human glioblastoma multiforme T98G cells. White arrows show the co-localized eIF1A and Ago2. (c) eIF1A interacts with Ago2. Human eIF1A was transiently overexpressed in stable HEK293 cells stably expressing Agos (1~4). Immunoprecipitation assays were performed with anti-eIF1A (monoclonal) antibody and immuno blotting (IB) with anti-Agos antibodies in the upper panel and anti-eIF1A antibodies in the bottom panel.

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• WB

CiteAb

Western blot - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Western Blotting using Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free, ab57113. Publication image from Gygi, S. P. et al., 2015, Nat Commun, 26018492. Legend direct from paper.

eIF1A binds to MID-domain of Ago2 in a RNA-binding-independent manner(a) Human eIF1A interacts with the MID fragment of human Ago2. Recombinantly expressed and Benzonase-treated Ago2 fragments and recombinantly expressed GST-eIF1A were used for GST-pull down analyses followed by Western blotting with anti-His antibodies (IB, immuno-blotting). The arrow shows that the MID fragment interacts with eIF1A. (b) The globular domain (GD) of eIF1A interacts with Ago2. Recombinantly expressed and Benzonase-treated GST, GST-eIF1A and mutants (ND, 25 ~ 144aa; CD, 1 ~ 114aa; GD, 25 ~ 114aa) were used for GST-pull down assays with cell lysates of HEK293 stably expressing Ago2. (c) 1H-15N TROSY-HSQC spectrum of 15N-eIF1A titrated with the sumo-MID (432 ~ 575aa). The titration of RNA-free-sumo-MID leads to broadening of RNA free 15N-eIF1A resonances (molar ratio of 1 : 1). Inserted frames show the broadening of resonances of V55, K56 and K67 residues in eIF1A upon MID titration. (d) 1H-15N TROSY-HSQC spectrum of 15N-Sumo-MID titrated with eIF1A. The titration of eIF1A (molar ratio of 1 : 1) results in broadening 1H-15N TROSY-HSQC crosspeaks coming from the 15N-MID domain, but not from 15N-Sumo (SMT3) tag. Inserted frames show the broadening of resonances of residues from MID-domain, but not from SMT3-Tag (SUMO-tag), upon eIF1A titration.

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• WB

CiteAb

Western blot - Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free (AB57113)

Western Blotting using Anti-Argonaute-2 antibody [2E12-1C9] - BSA and Azide free, ab57113. Publication image from Gygi, S. P. et al., 2015, Nat Commun, 26018492. Legend direct from paper.

eIF1A augments Ago2-mediated RNAi(a) Sequence of the human HMGA2 3′-UTR fragment mutant with two native let-7 target sites. Filled circles : Watson-Crick complementarity; open circles : non-Watson-Crick complementarity. Mutated nucleotides are shown in blue and wild type nucleotides were underlined (to neutralize site 2 and to reduce self-complementarity). (b) In vitro effects of eIF1A on Ago2-mediated RNA cleavage. eIF1A or the indicated mutants were incubated with Ago2, synthetic let-7a and 5′-32P end-labeled HMGA2 3′-UTR mutants. RNAs were analyzed by Northern blot and autoradiographs analyses. (c) Statistical analyses of three independent in vitro assays in (b). (d) Western blot assays of effects of eIF1A and eIF1A (K56A) mutant on GFP-let-7 reporter expression. Ago2, eIF1A or eIF1A (K56A) were transiently overexpressed in HEK293 cells stably expressing reporter GFP-let-7. (e)The expression ratio between Tubulin and GFP reporter in the same gel were calculated. Intact (100%) RISC activity was considered in control cells. The relative RISC activities under overexpression conditions of the indicated genes or mutants were obtained by comparison with the control. (f) Relative GFP reporter mRNAs in (d) by quantitative real-time PCR (qPCR). (g) Western blot assays of zebrafish c-Myb in the indicated zebrafish groups at 28 h.p.f (control, eif1axb MO (62.5µM, 1nl), eif1axb MO (62.5µM, 1nl) plus human eIF1A (heIF1A) (mRNA, 1µg/µl, 1000pl), and eif1axb MO (62.5µM, 1nl) plus heIF1A (K56A) mutant (mRNA, 1µg/µl, 1000pl)). Proteins were extracted from >10 zebrafish per group. (h) Relative c-Myb mRNAs in each group in (g) by qPCR. Total RNAs were extracted from >10 zebrafish per group. (i) Taqman PCR assays of relative zebrafish miR-126 (dre-miR-126) in each group in (g). Total RNAs were extracted from >10 zebrafish per group. In all statistical comparisons, three independent experiments were performed (mean ± s.d., n = 3 experiments, Student’s t-test). *** reports p < 0.01.

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