Anti-hnRNP A1 antibody [9H10]

• WB

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Western blot - Anti-hnRNP A1 antibody [9H10] (AB5832)

All lanes:

Western blot - Anti-hnRNP A1 antibody [9H10] (ab5832) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 38 kDa

Observed band size: 37 kDa,43 kDa

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Exposure time: 30s

• Flow Cyt

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Flow Cytometry - Anti-hnRNP A1 antibody [9H10] (AB5832)

Overlay histogram showing Jurkat cells stained with ab5832 (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 (ab5832, 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 IgG2b [PLPV219] (ab91366, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP A1 antibody [9H10] (AB5832)

ab5832 staining human lung. Staining is localized to the nucleus.
Left panel : with primary antibody at 1 ug/ml. Right panel : isotype control.
Sections were stained using an automated system (Dako PT Link), at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer, citrate pH 6.0. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• IP

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Immunoprecipitation - Anti-hnRNP A1 antibody [9H10] (AB5832)

hnRNP A1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to hnRNP A1 (ab5832) 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 ab5832.
Secondary : Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band : 37kDa : hnRNP A1; 42kDa : We are unsure as to the identity of this extra band.

All lanes:

Immunoprecipitation - Anti-hnRNP A1 antibody [9H10] (ab5832)

Predicted band size: 38 kDa

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• WB

AbReview21447****

Western blot - Anti-hnRNP A1 antibody [9H10] (AB5832)

Knockdown for 72 hours.

All lanes:

Western blot - Anti-hnRNP A1 antibody [9H10] (ab5832) at 1/1000 dilution

Lane 1:

Mouse NSC34 whole cell lysate at 10 µg with control siRNA

Lane 2:

Mouse NSC34 whole cell lysate at 10 µg with hnRNP A1 siRNA

Secondary

All lanes:

IRDye® 700DX-conjugated Donkey anti-Mouse IgG at 1/3000 dilution

Predicted band size: 38 kDa

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Exposure time: 1min

This image is courtesy of an anonymous Abreview.

• WB

CiteAb

Western blot - Anti-hnRNP A1 antibody [9H10] (AB5832)

Western Blotting using Anti-hnRNP A1 antibody [9H10], ab5832. Publication image from Buratti, E. et al., 2013, Nucleic Acids Res, 23863836. Legend direct from paper.

The first 44 nt of the Ron ESS element mediate splicing inhibition activity and interact with hnRNP A1. (A) Sequence of the previously identified 84 nt ESS. The position of SIL-I, SIL-II and SIL-III elements is indicated. Numbers refer to the cDNA sequence. (B) The entire ESS, SIL-I, SIL-II, SIL-III and ESE elements were cloned into the second exon of the human β-globin (Hβ) minigene. Minigenes were transfected into KATOIII cells; 24 h later RNAs were analyzed by RT-PCR with primer set β-globin_A and D (8) to detect both spliced (mRNA) and unspliced transcripts (pre-mRNA). Total (pre-mRNA plus mRNA, primers β-globin_A and Hbeta/CONTROL) and Spliced (mRNA, primers β-globin_A and Hbeta/DWsplicing) molecules were quantified by using qRT-PCR analysis; the ratio between Spliced and Total was plotted in the histogram by using the Hβ vector as a reference value. (C) Putative binding sites for hnRNP A1 (Box1) and SRSF6 (Box2) identified within SIL-I element by bioinformatics analysis. (D) In vitro pull-down assay with HeLa nuclear extracts (NE) and with a SIL-I riboprobe containing at its 3′ end the binding site for splicing factor TDP-43, as an internal control of pull-down efficiency. Materials were analyzed by western blotting with antibodies for indicated proteins.

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• WB

CiteAb

Western blot - Anti-hnRNP A1 antibody [9H10] (AB5832)

Western Blotting using Anti-hnRNP A1 antibody [9H10], ab5832. Publication image from Buratti, E. et al., 2013, Nucleic Acids Res, 23863836. Legend direct from paper.

HnRNP A1 binding to the Ron ESS interferes with SRSF1 binding to the ESE element. (A) The panel on the left shows the sequence of the wild type SIL-I probe and its different mutated versions. Right panel : KATOIII cells were transfected with the indicated Hβ vectors and RNAs were analyzed by RT-PCR as in Figure 1B. (B) In vitro pull-down assay with HeLa nuclear extracts and the indicated RNA probes : WT Ron ESS (84 nt), ESS mutated in Box2 (mutB = TGCGGC replaced with ATCGAC), the Ron ESE (67 nt), ESE plus ESS elements (151 nt) WT or mutated in Box2. Affinity bound proteins were analyzed by western blotting with the indicated antibodies. (C) RNA immunoprecipitation (IP) following UV cross-linking using anti-hnRNP A1 mAb 9H10 or IgG on HeLa cells. The abundance of Ron exon 12 or, as control, 7SL, 5S and tRNA-Leu enrichments were measured by qRT-PCR.

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• WB

CiteAb

Western blot - Anti-hnRNP A1 antibody [9H10] (AB5832)

Western Blotting using Anti-hnRNP A1 antibody [9H10], ab5832. Publication image from Buratti, E. et al., 2013, Nucleic Acids Res, 23863836. Legend direct from paper.

HnRNP A1 inhibits the production of the δRon transcript. (A) RT-PCR analysis of the splicing profile of endogenous Ron transcripts in HEK-293, HeLa and KATOIII cells with primers 2507 and 2991 (8). The histogram shows the qRT-PCR analysis of the total hnRNP A1 transcripts (primers hnRNPA1for and rev). On the right, western blot analysis of hnRNP A1 andα-tubulin protein levels in the indicated cell lines. (B) Scheme of the Ron minigene p2507–2991 (8) and of a derivative mutated in the ESS sequence (pMut-SIL-I). (C) HEK-293 and (D) KATOIII cells were transfected with the indicated minigenes and RNAs analyzed with primers 2507 and BGHrev (8); t-Ron and t-δRon indicate minigene transcripts generated by inclusion and skipping of exon 11, while histograms show the t-δRon/t-Ron measured in three independent experiments. (E) HeLa cells were transfected with GFP-hnRNP A1 or with the empty vector (‘Vector’). The expression of the overexpressed protein was verified by western blotting with anti-GFP and anti-α-tubulin antibodies. On the right, p2507–2991 and pMut-SIL-I minigenes were co-transfected into HeLa cells with the GFP-tagged hnRNP A1 or the empty vector. Total RNAs were analyzed by RT-PCR as in Figure 3B.

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• WB

CiteAb

Western blot - Anti-hnRNP A1 antibody [9H10] (AB5832)

Western Blotting using Anti-hnRNP A1 antibody [9H10], ab5832. Publication image from Buratti, E. et al., 2013, Nucleic Acids Res, 23863836. Legend direct from paper.

HnRNP A1 affects the MET program. (A) HEK-293 cells not treated (NT) or transfected with hnRNP A1 (A1) or with control siRNAs (Ctr). Total cell extracts were probed by western blotting with the following antibodies : anti hnRNP A1 (9H10), anti-hnRNP H, anti-SRSF1 (mAb96) and anti-α-tubulin; the three histograms on the right show the relative level of hnRNP A1, SRSF1 and hnRNP H transcripts quantified by qRT-PCR. (B) The same cells (not treated or transfected with siRNA oligos) were analyzed in (i) RT-PCR to determine the splicing profile of the endogenous Ron transcripts as in Figure 3A and (ii) qRT-PCR for the expression level of the indicated EMT markers. Cells morphologies were examined under a phase-contrast microscope (objective 10x with additional magnification 1.6x). (C) HeLa cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) with an efficiency of ∼60%; RNAs were analyzed with RT-PCR (as in Figure 3A) to determine the splicing profile of Ron exon 11 and cell extracts were analyzed in western blotting to assess the abundance of the indicated proteins and the expression of T7-hnRNP A1. The same cells were also analyzed in qRT-PCR for the expression levels of selected EMT markers. Panel on the right shows the results of the wound healing assay. Transient transfected HeLa cells were grown to confluence and wounded by dragging a 200 µl pipette tip through the monolayer (0 h); cell migration was measured at designated times (5, 10 and 24 h) after wounding. (D) MDA-MB-435S cells stably transfected with T7-SRSF1 (Cl.SF2) or with the empty vector (Cl.Vector) (29). Cl.SF2 and Cl.Vector cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) as indicated; total cell extracts were analyzed in western blotting to assess the level of the indicated proteins. (E) Total RNAs from the same cells were analyzed by RT-PCR (as in Figure 3A); the histogram below shows the δRon/Ron ratio. Expression level of the epithelial marker E-cadherin in the different cells as assessed by qRT-PCR is also shown.

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