Anti-ENO1 + ENO2 + ENO3抗体

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

ICC/IF image of ab53025 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53025, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

Immunohistochemical analysis of paraffin-embedded human brain tissue using ab53025 at 1/50-1/100 dilution. Left : without immunizing peptide; Right : with immunizing peptide

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

ab53025 staining NSE in mouse brain tissue section by Immunohistochemistry (PFA-perfussion fixed frozen tissue sections). Tissues were collected from mice in each group at 10 weeks. Mice were euthanized, the brains were exposed and removed from the body. Then the brain was cut, on ice and fixed in 4% polyformaldehyde or glutaraldehyde solution for histological examination. The primary antibody was used at 1/75 dilution in PBS and incubated with sample for 2 hours at 37°C. A HRP-conjugated secondary antibody was used at for 1hr at 37°C in a humidity box.

Image from Yu Yang. et. al. Mol. Cell. Proteomics, Mar 2009 (Fig 6).

• WB

Unknown

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

All lanes:

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody (ab53025) at 1/500 dilution

Lane 1:

Extracts from HepG2 cells with no immunizing peptide

Lane 2:

Extracts from HepG2 cells with immunizing peptide

Predicted band size: 47 kDa

Observed band size: 47 kDa

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• WB

Lab

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

Western blot : Anti-ENO1 + ENO2 + ENO3 antibody ab53025 staining at 1/1000 dilution, shown in green; Mouse anti-6x HisTag (ab18184) loading control staining at 1/20,000 dilution, shown in magenta.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody (ab53025) at 1/1000 dilution

Lane 1:

ENO1 Recombinant Protein (His Tag) at 0.2 µg

Lane 2:

ENO2 Recombinant Protein (His Tag) at 0.2 µg

Lane 3:

ENO3 Recombinant Protein (His Tag) at 0.2 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 47 kDa

Observed band size: 47 kDa

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• WB

CiteAb

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

Western Blotting using Anti-NSE antibody - Neuronal Marker, ab53025. Publication image from Yin, Y. et al., 2019, Mol Cancer, 30657058. Legend direct from paper.

N-Myc confers LNCaP cells ADT resistance and C4–2 cells Enzalutamide resistance. a Immunoblots to show the decreased AR and PSA expression but increased expression of neuroendocrine markers, CgA and NSE, in both N-Myc overexpressing LNCaP and C4–2 cells. GAPDH was used as a loading control. b Growth curve for LNCaP/Vec vs. LNCaP/N-Myc and C4–2/Vec vs. C4–2/N-Myc cells cultured in regular medium and cell number was counted at the indicated time points. All the numbers were normalized to day 0. c Overexpression of N-Myc increased colony formation in both LNCaP and C4–2 cell lines but had a more profound effect on LNCaP cells. Upper panels showed representative images and lower panels were quantifications. d Growth curve for LNCaP/Vec and LNCaP/N-Myc cells cultured in charcoal-stripped medium (ADT) and cell numbers was counted at the indicated time points. All the numbers were normalized to day 0. e LNCaP/Vec and LNCaP/N-Myc cells were injected into the flanks of nude mice. Tumor volume was measured twice a week at indicated time points (n = 4 for each group). f Castration was initiated when tumor volume reached ~ 200mm3 and set as day 0. Tumor volume was measured twice a week and relative volume was reported (n = 4 for each group). g Dose response of Enzalutamide at 72 h using MTS cell viability assay for C4–2/Vec and C4–2/N-Myc cells. *p < 0.05, **p < 0.005

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• WB

CiteAb

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody (AB53025)

Western Blotting using Anti-NSE antibody - Neuronal Marker, ab53025. Publication image from Poutanen, M. et al., 2020, Nat Commun, 31959826. Legend direct from paper.

Androgen-deprivation upregulates SPINK1 in NE-transdifferentiated PCa cells.a Bar graph showing SPINK1 expression (top) and heatmap of AR-signaling associated genes including SPINK1 in long-term androgen deprived (AD) LNCaP cells (GSE8702). b Representative phase-contrast images of androgen-deprived LNCaP cells (LNCaP-AI). Red arrow-heads indicate neurite outgrowth. c QPCR data showing relative expression of SPINK1 and KLK3 using same cells as b. d Immunoblot assay for SPINK1, PSA, SYP, ENO2, SOX2 and REST using same cells as in b. e Immunostaining for SPINK1 using same cells as in b. f Schematic representation of NE-transdifferentiation (NE-td) using doxycycline (dox)-inducible AR-V7 overexpressing LNCaP cells subjected to androgen deprivation (AD) with or without induction (40 ng/ml) at day 10 and cultured upto 30 days. g QPCR data showing relative expression of SPINK1 and KLK3 using same cells as f. h Immunoblot assay for AR, AR-V7, SPINK1, and PSA using same cells as f. i QPCR data showing relative expression of SYP, NCAM1, ENO2 and CHGA using the same cells as f. j Schema describing generation of LNCaP-AI-shSPINK1 and LNCaP-AI-shSCRM cells by subjecting stable LNCaP-shSPINK1 and LNCaP-shSCRM cells to androgen deprivation (AD) for 30 days. k Representative images for the neurite outgrowths in LNCaP-AI-shSCRM cells and LNCaP-AI-shSPINK1 as j (top). QPCR data showing relative expression of SPINK1 (bottom, left) and measurement of neurite outgrowth (bottom, right). l Immunoblot analysis for SPINK1, E-Cad, VIM, and N-Cad expression using same cells as j. m Same as in l, except phospho (p) and total (t) AKT, SYP and ENO2 expression. n Representative IHC images for SPINK1 in SPINK1-negative (SPINK1−, top) and SPINK1-positive (SPINK1+, bottom) PCa tumor cores of the VPC tissue microarray (scale bar = 200 µm). Bar plot showing percentage cases of SPINK1 in untreated (n = 33) and neoadjuvant-hormone therapy (NHT) treated patients (n = 55). Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels b, e, k scale bar represents 20 µm. For panel c two-way ANOVA, Dunnett’s multiple-comparisons; g, i two-way ANOVA, Sidak’s multiple-comparisons test; k one-way ANOVA, Dunnett’s multiple-comparisons test were applied. ∗P ≤ 0.05 and ∗∗P ≤ 0.001. Source data for d, h, l, m are provided as a Source Data file.

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