Anti-Sortilin/NT3 antibody

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody (AB16640)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Sortilin/NT3 knockout HAP1 cell lysate (20 μg)
Lane 3 : NIH/3T3 cell lysate (20 μg)
Lane 4 : 293T cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab16440 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab16640 was shown to specifically react with Sortilin/NT3 in wild-type HAP1 cells. No band was observed when Sortilin/NT3 knockout samples were examined. Wild-type and Sortilin/NT3 knockout samples were subjected to SDS-PAGE. ab16640 and ab8245 (loading control to GAPDH) were diluted 1 μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody (ab16640)

Predicted band size: 92 kDa

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• IHC-FoFr

Collaborator

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Sortilin/NT3 antibody (AB16640)

Immunofluorescent staining for Sortilin/NT3 in rat cortical neurons using ab16640. The staining is cytoplasmic and punctate of many cells such as cortical neurons. The image was taken with a X20 objective.

Protocol details : Rats were intracardially perfused with 4% paraformaldehyde. Whole brain tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT and then cut on cryostat (30μm coronal sections). IHC was perfored in free floating with fixed tissues (rat brain sections). Primary antibody was incubated overnight at 1/3000 at room temperature

This image is courtesy of Sophie Pezet, Univ London Kings Coll, United Kingdom

• WB

Unknown

Western blot - Anti-Sortilin/NT3 antibody (AB16640)

Lanes 1- 2 : Merged signal (red and green). Green - ab16640 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab16640 was shown to react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264772 (knockout cell lysate ab257696) was used. Wild-type HeLa and SORT1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab16640 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody (ab16640) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-sort1-sortilin-nt3-knockout-hela-cell-lysate-ab257696'>ab257696</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 100 kDa

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• WB

Project

Western blot - Anti-Sortilin/NT3 antibody (AB16640)

Lane 1:

Marker

Lanes 2 - 4:

Western blot - Anti-Sortilin/NT3 antibody (ab16640) at 1 µg/mL

Lane 2:

Human Brain tissue lysate at 20 µg

Lane 3:

Mouse Brain tissue lysate at 20 µg

Lane 4:

Rat Brain whole cell lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution

Predicted band size: 92 kDa

Observed band size: 31 kDa

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• WB

CiteAb

Western blot - Anti-Sortilin/NT3 antibody (AB16640)

Western Blotting using Anti-Sortilin/NT3 antibody, ab16640. Publication image from Jauberteau, M. O. et al., 2017, Nat Commun, 29084952. Legend direct from paper.

EGF promotes the EGFR—sortilin interaction. a A549 cells grown in complete cell culture media were stimulated or not with EGF (50 ng/mL) for 30 min. Immunoprecipitations (IP) were performed using anti-EGFR antibody, and the immunocomplexes were immunoblotted (IB) using anti-sortilin antibody (top). In parallel, immunoblots for P-EGFR, EGFR, sortilin, P-ERK, and ERK were performed on whole-cell lysates (WCL); the isotypic lane Immunoglobulin G (IgG) represents the IP control. b Proximity ligation assays (PLA) were performed on A549 cells, non-stimulated or stimulated with EGF (50 ng/mL) for 2, 5, 15, and 30 min. Red spots indicate sites of proximity ligation assay amplification, reflecting the EGFR—sortilin interaction (white arrows). Scale bar, 10 µm. c Quantification of PLA time course, in comparison with non-stimulated cells. d A549 cells were stimulated or not with EGF (50 ng/mL) for 30 min, and then co-immunolabeled for EGFR and markers of the early endosome (Rab5), the late endosome/lysosome (LAMP2) and the trans-Golgi network (TGN46). For sortilin labeling, A549 cells were transiently transfected with sortilin-GFP to adapt a set of functional antibodies, and then immunolabeled for the same markers. Scale bar, 5 µm. e Quantitative analysis of EGFR or sortilin-GFP co-localization with the aforementioned organelle-specific markers. f A549 cells were transiently transfected with sortilin-GFP, and then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were fixed and co-immunolabeled for EGFR and Rab5. Scale bar, 10 µm. All values represent means ± SD, Student’s t-test *P < 0.05; ***P < 0.001. Each experiment has been repeated at least three times

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