- 44-786G - Invitrogen Antibodies EGFR antibody

Submitted references Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Minutti CM, Drube S, Blair N, Schwartz C, McCrae JC, McKenzie AN, Kamradt T, Mokry M, Coffer PJ, Sibilia M, Sijts AJ, Fallon PG, Maizels RM, Zaiss DM
Immunity 2017 Oct 17;47(4):710-722.e6

Tumor endothelial cells promote metastasis and cancer stem cell-like phenotype through elevated Epiregulin in esophageal cancer.

Sun L, Pan J, Yu L, Liu H, Shu X, Sun L, Lou J, Yang Z, Ran Y
American journal of cancer research 2016;6(10):2277-2288

Molecular basis for multimerization in the activation of the epidermal growth factor receptor.

Huang Y, Bharill S, Karandur D, Peterson SM, Marita M, Shi X, Kaliszewski MJ, Smith AW, Isacoff EY, Kuriyan J
eLife 2016 Mar 28;5

Fyn inhibition rescues established memory and synapse loss in Alzheimer mice.

Kaufman AC, Salazar SV, Haas LT, Yang J, Kostylev MA, Jeng AT, Robinson SA, Gunther EC, van Dyck CH, Nygaard HB, Strittmatter SM
Annals of neurology 2015 Jun;77(6):953-71

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Extracts of A431 cells unstimulated (1) or stimulated with 200 ng/mL EGF for 15 minutes (2-5), were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of EGFR [pY992] was performed using 90% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at RT. The cells were labeled with Phospho-EGFR (Tyr992) Mouse Polyclonal Antibody (Product # 44-786G) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membranous localization. Panel e represents cells treated with antagonist, Afatinib (1µM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing reduced Phospho-EGFR staining. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of EGFR (pY992) showing staining in the cytoplasm and membrane of paraffin-embedded human hepatocellular carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a EGFR (pY992) Rabbit Polyclonal Antibody (Product # 44-786G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of EGFR (pY992) showing staining in the cytoplasm and membrane of paraffin-embedded human lung adenocarcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a EGFR (pY992) Rabbit Polyclonal Antibody (Product # 44-786G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of EGFR [pY992] was done on A549 cells treated with EGF (200ng/ml, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with EGFR [pY992] Rabbit Polyclonal Antibody (44786G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor¨ 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8. Effect of Domain IV mutations on phosphorylation of EGFR and two effector proteins. Wild-type (WT) or mutant EGFR was transfected into HEK293T cells, and phosphorylation was measured using FACS. The EGFR mutants are: II/KK (I545K, I556K); VEN/ERR (V526E, E527R and N528R); TN/RR (T548R and N554R). ( A ) Phosphorylation of a proximal site (Tyr 992) in EGFR. The bar graph shows the average phosphorylation level from cells expressing an intermediate level of EGFR, selected based on the whole range of EGFR expression in the FACS analysis, with (orange) and without (blue) the addition of EGF. ( B ) As in Panel A, for a distal site (Tyr 1173) in EGFR. ( C ) As in Panels A and B, for phosphorylation of PI3K. In addition to two mutations in Domain IV (II/KK and VEN/ERR), the phosphorylation levels for a kinase-dead EGFR variant (D813N) are shown. ( D ) Phosphorylation of ERK. The levels of phosphorylated ERK (pERK) show a bimodal distribution, with cells tending to have either low or high levels of pERK. The data are best represented as a histogram of cell numbers with different levels of pERK, as described ( Kovacs et al., 2015b ). Note that the response of two Domain IV mutants (II/KK and VEN/ERR) resembles that of the wild-type EGFR. For comparison, data for kinase-dead EGFR (D813N) show a reduced population of cells with high levels of pERK. DOI: http://dx.doi.org/ Figure 8--figure supplement 1. Transmembrane helix mutations. Effect on PI3K and ERK phosphorylation of

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 AREG Induces EGFR Phosphorylation at Tyr-992, which Allows for the Interaction between T1/ST2 and EGFR WT (gray) and Areg -/- (purple) mice were infected with H. polygyrus , and on day 14 post infection, mLN were harvested. (A and B) mLN cells were stimulated with rIL-33, anti-CD3, or media, and the IL-13 (A) and p-ERK (B) expression was determined by intra-cellular staining and flow cytometry analysis. (C) MHCII-deficient mice were infected with H. polygyrus and 7 days post-infection received flow cytometry-sorted CD4 + T cells derived from mLN of naive or H. polygyrus -infected WT, Egfr fl/fl xCd4-cre , or Areg -/- mice. Worm burden and egg counts were determined 2 weeks post infection. (D-F) mLN cells were stimulated with rIL-33, rAREG, both, or media only, and EGFR p-Y1068 (D), p-ERK (E), and IL-13 (F) expression was determined by flow cytometry analysis. (G) EGFR phosphorylation at position Y992 on CD4 + T cells derived from mLN of WT or Areg -/- H. polygyrus -infected mice in the presence or absence of rAREG. (H) HEK293T cells were transfected as indicated with T1/ST2 and the IL-1RacP in combination with WT EGFR or EGFR Y992F mutant. Subsequently, the cell lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot. (I) mLN cells wer

44-786G

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