Anti-EEA1 抗体 - Early Endosome Marker

• WB

Unknown

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

Fluorescence detection of secondary antibody.

All lanes:

Western blot - Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) nuclear lysate at 20 µg

Lane 2:

HeLa whole cell lysate at 20 µg

Lane 3:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 5:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Secondary

All lanes:

Alexa Fluor anti rabbit at 1/50000 dilution

Predicted band size: 162 kDa

Observed band size: 100 kDa,180 kDa,41 kDa,50 kDa

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• WB

Ap

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin (Lanes 1-3) or 3% Milk (Lanes 4-6) before being incubated with ab2900 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

All lanes:

Western blot - Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1 µg/mL

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Lane 5:

HEK-293 whole cell lysate at 20 µg

Lane 6:

NIH 3T3 whole cell lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG H&L (HRP) at 1/50000 dilution

Predicted band size: 162 kDa

Observed band size: 100 kDa,180 kDa

true

Exposure time: 30s

• WB

Lab

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : EEA1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : NIH3T3 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab2900 observed at 162 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab2900 was shown to recognize EEA1 in wild-type HAP1 cells as signal was lost at the expected MW in EEA1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and EEA1 knockout samples were subjected to SDS-PAGE. ab2900 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-EEA1 antibody - Early Endosome Marker (ab2900)

Predicted band size: 162 kDa

false

• WB

Unknown

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

Lane 1 - 2 : EEA1 antibody - Early Endosome Marker (ab2900) at 1/500 dilution, HEK293 Whole Cell lysate at 20 ug Lane 1 : as above Lane 2 : EEA1 peptide (ab14946) at 1 ug Secondary Goat polyclonal to Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution Performed under reducing conditions. Predicted band size : 160kD Observed band size : 180kD (why is the actual band size different from the predicted?)

All lanes:

Western blot - Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1/500 dilution

Lane 1:

HEK293 Whole Cell lysate at 20 µg

Lane 2:

HEK293 Whole Cell lysate at 20 µg with Human EEA1 peptide (ab14946)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 162 kDa

Observed band size: 180 kDa

false

• WB

Project

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

The Western blot shows that ab14944 reacts strongly with mouse 3T3 and CHO cell lysates. Weak cross-reactivity is seen with Xenopus, mouse brain, mouse liver, rat brain and dog lysates. The antibody does not appear to cross-react with rat liver lysate.

All lanes:

Western blot - Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1 µg/mL

Lane 1:

Xenopus laevis lysate at 20 µg

Lane 2:

Mouse 3T3 cell lysate at 20 µg

Lane 3:

Mouse brain cell lysate at 20 µg

Lane 4:

Mouse liver cell lysate at 20 µg

Lane 5:

Rat brain cell lysate at 20 µg

Lane 6:

Rat liver cell lysate at 20 µg

Lane 7:

Dog lysate at 20 µg

Lane 8:

CHO 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-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 162 kDa

Observed band size: 100 kDa,180 kDa

false

Exposure time: 30s

• WB

CiteAb

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

Western Blotting using Anti-EEA1 antibody - Early Endosome Marker, ab2900. Publication image from Shi, M. et al., 2018, Nat Commun, 30478271. Legend direct from paper.

Starvation translocates TGN membrane proteins to endosomes. All cells are HeLa cells. a, b Furin loses its Golgi localization during starvation. Cells treated with indicated medium for 1 h and endogenous furin and Golgin-245 were stained. The fraction of Golgi-localized furin is quantified in b. c The recovery of Golgi localization of furin after supplying nutrient. After starvation in HBSS for 2 h, cells were treated with DMEM for indicated time and stained as in a. d Kinetics of Golgi-localized furin-GFP during HBSS and subsequent DMEM treatment. Cells expressing furin-GFP were first starved in HBSS for 2 h and subsequently stimulated by DMEM for 2 h. At indicated time, cells were stained for endogenous Giantin and the fraction of Golgi-localized furin-GFP is quantified. e, f Nutrient starvation significantly reduces the Golgi localization of CD8a-furin. Cells transiently expressing CD8a-furin were treated by indicated medium for 2 h and stained as in e. The fraction of Golgi-localized CD8a-furin is quantified in f. g The translocation of CD8a-furin to the endosome during nutrient starvation. Cells transiently expressing indicated constructs were treated with HBSS for 2 h and stained. Boxed regions are enlarged at the upper right corner. Arrows indicate colocalization. h, i The endosomal pool of CD8a-furin increases during nutrient starvation. Similar results have been observed in four independent experiments. Cells stably expressing CD8a-furin were treated with HBSS for 2 h or with additional 20 min treatment of DMEM. Lysates were subjected to sucrose gradient centrifugation to separate organelles. 20 fractions were collected and immunoblotted for CD8a-furin and markers. Percentages of CD8a-furin distributed in the endosomal (fractions 1–5) and TGN pool (fractions 10–16) are quantified in i. b, d, and f are representative results from three independent experiments. Complete, complete medium, n, the number of cells analyzed; error bar, mean ± s.e.m.; scale bar, 10 µm. P values were from t test (unpaired and two-tailed). ***P ≤ 0.0005

false

• WB

CiteAb

Western blot - Anti-EEA1 antibody - Early Endosome Marker (AB2900)

Western Blotting using Anti-EEA1 antibody - Early Endosome Marker, ab2900. Publication image from Choquet, D. et al., 2022, Nat Commun, 35115539. Legend direct from paper.

Proteomic profiling of the autophagic cargo during NMDAR-LTD.a–d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p-values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t-test (GluA1,N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a–d and g are in kDaltons (kD).

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