Anti-EGFR (phospho Y1086) antibody [Y39]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

Immunofluorescent analysis of EGFR (phospho Y1086) expression in A431 cell culture, using 1/100 ab32086.

• WB

Unknown

Western blot - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

All lanes:

Western blot - Anti-EGFR (phospho Y1086) antibody [Y39] (ab32086) at 1/10000 dilution

Lane 1:

Untreated A431 cell lysate

Lane 2:

A431 cell lysate, treated with EGF

Predicted band size: 134 kDa

Observed band size: 150 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma cell line) cells labeling EGFR (phospho Y1086) with ab32086 at 1/100 3 μg/ml. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 a AlexaFluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1000 2 μg/ml. Cells were counterstained with ab195889 anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 2.5 μg/ml. Nuclear stain was DAPI (blue).

The green staining on the membrane was increased in the EGF (100ng/ml 10min) treated A431 cells when compared with A431 cells without treatment. After LP treatment the green signaling was obviously decreased.

For the pan antibody there was no great difference after EGF (100ng/ml 10min) or EGF (100ng/ml 10min) + LP treatment. The data showed mostly membranous staining.

• IP

Lab

Immunoprecipitation - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

Purified ab32086 at 1/20 dilution (1μg) immunoprecipitating EGFR in A431 treated with 100ng/mL EGF for 30min whole cell lysate.
Lane 1 (input) : A431 (Human epidermoid carcinoma epithelial cell) treated with 100ng/mL EGF for 30min whole cell lysate 10μg
Lane 2 (+) : ab32086 + A431 treated with 100ng/mL EGF for 30min whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32086 in A431 treated with 100ng/mL EGF for 30min whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 175 kDa

All lanes:

Immunoprecipitation - Anti-EGFR (phospho Y1086) antibody [Y39] (ab32086)

Predicted band size: 129 kDa,134 kDa

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• WB

Lab

Western blot - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-EGFR (phospho Y1086) antibody [Y39] (ab32086) at 1/3000 dilution

Lane 1:

Untreated A431 (Human epidermoid carcinoma cell line) whole cell lysates at 15 µg

Lane 2:

A431 (Human epidermoid carcinoma cell line) treated with 100 ng/ml EGF for 10 minutes whole cell lysates at 15 µg

Secondary

All lanes:

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

Predicted band size: 134 kDa

Observed band size: 170 kDa

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Exposure time: 10s

• Dot

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Dot Blot - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

Dot blot analysis of EGFR (phospho Y1086) phospho peptide (Lane 1) EGFR non-phospho peptide (Lane 2) labeling EGFR (phospho Y1086) with ab32068 at a dilution of 1/1000. Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 5 seconds.

• WB

CiteAb

Western blot - Anti-EGFR (phospho Y1086) antibody [Y39] (AB32086)

EGFR (phospho Y1086) western blot using anti-EGFR (phospho Y1086) antibody [Y39] ab32086. Publication image and figure legend from Hashikawa, K. I., Katamune, C., et al., 2017, Sci Rep, PubMed 28855649.

ab32086 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32086 please see the product overview.

Effects of p16INK4a on EGF receptor-mediated Ras activation in human keratinocytes. HaCaT cells were infected with retroviral vectors expressing Flag-tagged p16INK4a. (a) Protein levels of Flag-tagged p16INK4a, total Ras, and EGF receptor in HaCaT cells. Plus and minus indicate the infection with p16INK4a-Flag expressing vectors. Full-size images are presented in Supplementary Fig. 6. Data shown were confirmed in more than three independent experiments. (b) mRNA levels of inflammatory cytokines in cells infected or not infected with the p16INK4a-Flag expressing vectors. (Means ± s.e.m.; n = 6). **p < 0.01 significant difference control cells (t10 = -8.393, p < 0.001 for IL-6, t10 = -4.161, P = 0.002 for Ccl2, t10 = -3.975, P = 0.034 for Tnf-α; unpaired t-test, two-sided). (c) Protein levels of phosphorylated EGFR (right) and the amount of the active form of Ras (left) in cells infected or not infected with the p16INK4a-Flag expressing vectors. For right panel, western blot data were confirmed in three mice in each group. Full-size images are presented in Supplementary Fig. 7. For left panel, cells were incubated in serum-starved media for 24 hours and then stimulated with 200 ng/ml of EGF for 1 minute. Plus and minus indicate the introduction of the p16INK4a-Flag vector and the EGF treatment, respectively. Values of absorbance at 450 nm were obtained and normalized by total protein levels (Means ± s.e.m.; n = 4). *p < 0.05 significant difference between the two groups (F3,12 = 29.245, p < 0.001; ANOVA with Tukey-Kramer's post-hoc test).

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