Anti-proNGF 抗体 [EP1320Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NGF antibody [EP1320Y] (AB52918)

Immunofluorescence staining of U87-MG cells with purified ab52918 at a working dilution of 1 in 300 counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control purified ab52918 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

• WB

Supplier Data

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

All lanes:

Western blot - Anti-NGF antibody [EP1320Y] (ab52918) at 1/500 dilution

All lanes:

fetal thyroid tissue at 10 µg

Secondary

All lanes:

Goat anti rabbit IgG HRP antibody at 1/2000 dilution

Predicted band size: 27 kDa

Observed band size: 27 kDa

false

• WB

Supplier Data

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Blocking Buffer : 5% NFDM/TBST

Dilution Buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-NGF antibody [EP1320Y] (ab52918) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 20 µg

Lane 2:

Human fetal thymus lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Secondary

All lanes:

HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 27 kDa

Observed band size: 32 kDa

false

• WB

Supplier Data

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

The positive cell lines (Lanes 7-9) are consistent with the literature : PMID : 12944907, Fig1.

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-NGF antibody [EP1320Y] (ab52918) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Lane 3:

Human cerebellum lysate at 20 µg

Lane 4:

Mouse cerebellum lysate at 20 µg

Lane 5:

Rat cerebellum lysate at 20 µg

Lane 6:

Hela (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 8:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 27 kDa

Observed band size: 32 kDa

true

Exposure time: 20s

• WB

Supplier Data

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Blocking Buffer : 5% NFDM/TBST

Dilution Buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-NGF antibody [EP1320Y] (ab52918) at 1/1000 dilution

All lanes:

Mouse thyroid lysate at 20 µg

Secondary

All lanes:

HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 27 kDa

Observed band size: 32 kDa

false

• WB

Supplier Data

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Blocking Buffer : 5% NFDM/TBST

Dilution Buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-NGF antibody [EP1320Y] (ab52918) at 1/2000 dilution

All lanes:

Rat thyroid lysate at 20 µg

Secondary

All lanes:

HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 27 kDa

Observed band size: 32 kDa

false

• WB

CiteAb

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Western Blotting using Anti-NGF antibody [EP1320Y], ab52918. Publication image from Wang, P. et al., 2017, Nat Commun, 28440296. Legend direct from paper.

The antitumour and gene knockdown effects of GNC–siRNA complex in orthotopic tumours.(a) Scheme of siRNA treatment. Panc-1-luc cells were injected into the pancreas head of Balb/c nude mice to form orthotopic tumours. After 2 weeks, mice were divided into different groups. Mice received various formulations via tail veil injections for six times, and killed on day 28. (b) The changes of the mouse body weight during treatments. (c) In vivo whole-body bioluminescence images of mice on day 14 and day 28, which indicated the tumour size before and after siRNA treatment. Bioluminescence signal was a result from the interaction of luciferase from Panc-1-luc cells with D-luciferin injected into the mice before imaging. (d) Ex vivo bioluminescence images of orthotopic pancreatic tumours and tumour metastases into mesenteries on day 28. Yellow lines enclosed the locations of primary tumours in the pancreas. Scale bar, 1 cm. (e) Tumour images on day 28. Scale bar, 5 mm. (f) Quantification of in vivo bioluminescence to evaluate the primary tumours in mice on day 28. (g) Quantification of tumour metastases by the sum of ex vivo bioluminescence detected in the mesenteries on day 28. (h) Weight of the isolated tumours. (i) NGF mRNA and (j) NGF protein expression levels in orthotopic tumours. (k) Confirmation of RNAi-mediated mechanism of action with GNC–siRNA by 5′-RACE assay. A 2% agarose gel electrophotosis showed ∼370 bp RNA-induced silencing complex-mediated cleavage product for NGF siRNA in pancreatic tumours. Only tumours treated with GNC–siRNA complex showed the cleavage product at 370 bp. The left column was the DNA ladder with described molecular weights. (l) IF images of NGF protein (red) in the orthotopic tumours. (m) IF images of neurites (red) in the orthotopic tumours. The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 µm. (n) Quantification of the neurite density in the orthotopic tumours. Mean±s.d. (n=6–9 per group). Significant difference was from the saline control, *P<0.01, **0.01<P<0.05; Student's t-test.

false

• WB

CiteAb

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Western Blotting using Anti-NGF antibody [EP1320Y], ab52918. Publication image from Wang, P. et al., 2017, Nat Commun, 28440296. Legend direct from paper.

The antitumour and gene knockdown effects of GNC–siRNA complex in subcutaneous pancreatic tumours.Panc-1 cells were injected into the flank of Balb/c nude mice to form subcutaneous tumours. When the tumours reached about 5 mm in diameter, the animals received peritumoral injections of various formulations every 2 days for six injections. (a) Tumour growth curve during the treatments. The black arrows indicated the days of injection. (b) Ex vivo tumour image and (c) tumour weight at the end of experiment. Scale bar, 1 cm. (d) Expression level of NGF mRNA and (e) NGF protein level in subcutaneous tumours. (f) IF staining of neurites in tumour tissues with various siRNA treatments. Neurites were stained with neurofilament antibody (red), the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 µm. (g) Quantification of neurite density in the subcutaneous tumours by counting the neurite area positive to neurofilament antibody per unit area. Mean±s.d. (n=6 per group). Significant difference was from the saline control, *P<0.01, **0.01<P<0.05; Student's t-test.

false

• WB

CiteAb

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Western Blotting using Anti-NGF antibody [EP1320Y], ab52918. Publication image from Wang, P. et al., 2017, Nat Commun, 28440296. Legend direct from paper.

Characterization of GNC–siRNA in vitro.(a) Protection of siRNA against serum nucleases. Free siRNA and GNC–siRNA (100 nM siRNA) were incubated within 10% serum for multiple time points, and analysed by polyacrylamide gel electrophoresis. (b) Cellular uptake of free siRNA and GNC–siRNA into Panc-1 cells. siRNA was labelled with Cy5 dye (Cy5-siRNA), the Panc-1 cells were incubated with various Cy5-siRNA formulations for 1 h and observed by confocal microscope with a 633 nm laser excitation. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 µm. (c) Lysosomal escape of GNC–siRNA in Panc-1 cells. The lysosomes of cells were stained with LysoTracker Green for 1 h, and the Panc-1 cells were treated with GNC–Cy5-siRNA for 1 h. The cells were observed by confocal microscope over different time points. Scale bars, 20 µm. (d) Expression level of NGF mRNA in Panc-1 cells analysed by RT–PCR, the dotted line referred to the expression level of NGF mRNA in Panc-1 cells transfected with commercially available Lipofectamine 2000 transfection agent (Lipo2000-siRNA), which served as a positive control. Mean±s.d. (n=3). *P<0.01 compared with the nontreated control; Student's t-test. (e) Expression level of NGF protein in Panc-1 cells evaluated by western blotting. GNC binding with nsRNA was labelled as GNC–nsRNA and served as control siRNA.

false

• WB

CiteAb

Western blot - Anti-NGF antibody [EP1320Y] (AB52918)

Western Blotting using Anti-NGF antibody [EP1320Y], ab52918. Publication image from Wang, P. et al., 2017, Nat Commun, 28440296. Legend direct from paper.

The antitumour and gene knockdown effects of GNC–siRNA complex in orthotopic pancreatic PDX tumours.

(a) Scheme of the establishment of PDX tumour model. Patient-derived pancreatic tumours were trimmed, cut into fragments with similar sizes and transplanted into the pancreas head of the Balb/c nude mice. (b) Scheme of siRNA treatments. Two weeks after PDX tumour transplantation, mice were randomly divided into different groups and injected with various siRNA formulations through tail veil for six injections. The mice were killed on day 28. (c) The effect of different siRNA treatments on the changes of mouse body weight. (d) Representative images of the orthotopic PDX tumours in pancreas with associated spleen on day 28, tumours were indicated in red circles. (e) Tumour images and (f) tumour weight measured on day 28. Scale bar, 5 mm. (g) Representative images and quantification of tumour metastases into mesenteries. Tumour metastases were magnified and indicated by red circles. (h) NGF mRNA and (i) NGF protein level in the PDX tumours. (j) Representative images of the IF staining of NGF protein (red) in the PDX tumours. (k) Images of IF staining of neurites in PDX tumours. Neurites were stained with neurofilament antibody (red). The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 µm. (l) Quantification of neurite density in the PDX tumours. Mean±s.d. (n=5–6 per group). Significant difference was from the saline control, *P<0.01, **0.01<P<0.05; Student's t-test.

false