44-788G

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Provider product page for 44-788G

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [39]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Other assay [26]

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Product number: 44-788G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-EGFR (Tyr1068) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: Purified from rabbit serum by sequential epitope-specific chromatography, this product contains enough material for 10 mini-blots. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated epidermal growth factor receptor (EGFR). The final product is generated by affinity chromatography using an EGFR-derived peptide that is phosphorylated at tyrosine 1068. Positive controls used in western analysis were human epidermoid carcinoma (A431) cells +/- EGF.
Reactivity: Human, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

EGFR protein structure - 44-788G shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Inhibition of the phosphorylation of EGFR and downstream proteins by EGFR-TKIs in BaF3 cells harboring EGFR mutations The results of immunoblotting for Ba/F3 cells with EGFR exon 19 deletion, L858R, exon 19 deletion+T790M, and L858R+T790M are shown. The cells were treated with the indicated concentrations of EGFR-TKIs for 4 h. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. pEGFR, pAKT, and pERK indicate the phosphorylated form of EGFR, AKT, and ERK, respectively. Actin was used as a loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Inhibition of EGFR suppresses SP frequency and self-renewal. ( A ) H1650 and A549 cells revealed a significant decrease in SP frequency after treatment with 10 mug/ml EGFR-neutralizing antibodies for 5 days. ( B ) Frequency of SP cells in control antibody (1) and EGFR-neutralizing antibodies (2) treated cells. ( C ) Western blot analysis for ABCG2 and phospho-EGFR after EGFR-neutralizing antibody treatment. ( D ) SP frequency in NSCLC cells transfected with siRNA against EGFR or a control siRNA. ( E ) Expression of ABCG2 and EGFR in antibody treated cells. ( F and G ) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated per well from 1000 cells is plotted (mean +- SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. * represent the p value of

Supportive validation

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Experimental details: Figure 5 Inhibition of EGFR signaling downregulates Sox2 expression (A) H1650-SPAdh cells were treated with EGFR or Src inhibitors for 4 days and western blot analysis was performed for the indicated proteins. ( B ) H1650-SPAdh cells were treated with MEK or PI3K inhibitor for 4 days and levels of Sox2 and activation of Akt or ERK was evaluated by western blot. ( C ) A549 and H1975 cells were treated with MEK or PI3K inhibitor for 5 days and the frequency of SP cells were examined by SP analysis. Data represents the fold change in mean (+- SD) of SP frequency. ( D ) ABCG2 expression upon inhibitor treatment as detected by western blotting. ( E ) SP cell frequency in NSCLC cell lines transfected with specific siRNAs against Src or AKT. ( F ) ABCG2 expression in siRNA transfected cells as detected by western blotting. ( G and H ) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated from 1000 cells is plotted (mean +- SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. * represent the p value of

Supportive validation

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Experimental details: Fig 1 Characterization of HCC4006ER cells, which are highly resistant to erlotinib or the irreversible EGFR-TKI CL387,785. A, HCC4006 and HCC4006ER cells were treated for 72 hours with increasing concentrations of erlotinib or CL387,785. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. The error bars represent SEM of 3 independent experiments. B, HCC4006 and HCC4006ER cells were incubated for 24 hours at ~80-90% of cell confluence. Whole cell lysate of each cell line was collected and subjected to Proteome Profiler Human Phospho-RTK Array Kit to examine the phosphorylation levels of multiple RTKs. Detected phospho-RTKs on the array are circled. The spots in the four corners of the RTK array are positive controls. C, HCC4006 and HCC4006ER cells were incubated for 6 hours +- erlotinib (1 muM). Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, EGFR, pMET, MET, pHer3, Her3, PTEN, pAkt, Akt, pErk, Erk, and beta-actin.

Supportive validation

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Experimental details: Fig 2 Neither Her3 loss nor persistent activation of pAkt and pErk is essential for the resistance in HCC4006ER cells. A, HCC4006ER cells with stable GFP or Her3 overexpression (HCC4006ER-GFP and HCC4006ER-Her3 cells) as well as HCC4006 and the original HCC4006ER cells were treated for 72 hours with increasing concentrations of erlotinib. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. The error bars represent SEM of 3 independent experiments. B, HCC4006, HCC4006ER, HCC4006ER-GFP, and HCC4006ER-Her3 cells were incubated for 6 hours +- erlotinib (1 muM). Cell lysates were subjected to protein expression analysis with antibodies to Her3, pEGFR, EGFR, PTEN, pAkt, Akt, pErk, Erk, and beta-actin. C, HCC4006ER cells were treated for 72 h with increasing concentrations of erlotinib alone, BEZ235 alone, AZD6244 alone, or BEZ235 and AZD6244 in combination. HCC4006 cells were treated for 72 hours with increasing concentrations of erlotinib for 72 hours to plot a reference curve. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. The error bars represent SEM of 3 independent experiments. Combination index (CI) at IC50 dose of BEZ235 combined with AZD6244 was calculated by CompuSyn software. CI>1, CI = 1, and CI

Supportive validation

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Experimental details: Fig 6 Effects of knockdown of ZEB1 or overexpression of miR-200c on EMT markers and erlotinib sensitivity in HCC4006ER cells. A, HCC4006ER cells were plated at 3x10 5 per well in 6-well plate and transfected with ZEB1 or control siRNA at 5 nM as a final concentration. Cells were harvested for analysis at 96 hours post-transfection. Cell lysates were subjected to protein expression analysis with antibodies to ZEB1, E-cadherin, vimentin, and beta-actin. B, HCC4006ER cells were plated at 3x10 3 cells/ well in black wall 96-well plate, transfected with ZEB1 or control siRNA at 5 nM as a final concentration, and treated for 72 hours at 48 hours after siRNA transfection with increasing concentrations of erlotinib. HCC4006 cells were treated for 72 hours with increasing concentrations of erlotinib for 72 hours to plot a reference curve. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for erlotinib-untreated cells with control siRNA. Control siRNA or transfection reagent did not affect cell viability in HCC4006ER cells at this concentration. The error bars represent SEM of 3 independent experiments. C, HCC4006 and HCC4006ER cells were plated at 3x10 5 per well in 6-well plate. HCC4006ER cells were transfected with ZEB1 or control siRNA at 0.5 or 5 nM as indicated. HCC4006 cells and siRNA-transfected HCC4006ER cells were incubated for 6 hours +- erlotinib (1 muM) at 48 hours after siRNA transfection. Cell lysates were subjected to prot

Supportive validation

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Experimental details: Fig. 3 Effect of treatment with trastuzumab for 8 days on the expression profile of HER and pHER receptors. All gastric cancer cell lines were treated for 8 days with 10 ug/ml trastuzumab; afterwards, the expression of EGFR, HER2, HER3, HER4, pEGFR, pHER2, pHER3 and pHER4 was determined via Western blot analysis. Basal expression of HER receptors varied highly between the cell lines. No obvious correlation between basal HER/pHER receptor expression and/or changes in expression and responsiveness to trastuzumab could be observed

Supportive validation

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Experimental details: Fig. 4 Effect of treatment with cetuximab for 8 days on the expression profile of HER and pHER receptors. All gastric cancer cell lines were treated for 8 days with 10 ug/ml cetuximab; afterwards, the expression of EGFR, HER2, HER3, HER4, pEGFR, pHER2, pHER3 and pHER4 was determined via Western blot analysis. Only minor effects of the treatment on HER/pHER receptor profile were detected. No obvious correlation between basal HER/pHER receptor expression and/or changes in expression and responsiveness to cetuximab could be observed

Supportive validation

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Experimental details: Fig. 8 Effect of trastuzumab and concomitant ligand application on pEGFR and pHER2 levels in GSU and MKN45 cells. GSU and MKN45 cells were treated for 6 h with 10 ug/ml trastuzumab (Tra) and 15 ng/ml AREG (A) or 0.1 ng/ml EGF (E) or 0.4 ng/ml HB-EGF (H) or 0.75 ng/ml TGFalpha (T). Subsequently, pEGFR and pHER levels were determined by Western blot analysis. a Shows representative experiments; b shows results of densitometric measurements of three independent experiments. Treatment with trastuzumab induced the levels of both proteins in GSU cells but not in MKN45 cells. Concomitant application of the ligand suppressed this effect in GSU cells (significant suppression for pEGFR and HB-EGF). p values at significance levels of

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 The sensitivities of lung cancer cell lines to EGFR-TKIs (A) MTS assays of PC-9, PC9-ER, and H1975 cells exposed to the indicated EGFR-TKIs. Data points represent the mean +- standard deviation. (B) PC-9, PC9-ER, and H1975 cells were treated with the indicated concentrations of EGFR-TKIs for 4 h prior to immunoblotting for the phosphorylated (p) and non-phosphorylated forms of EGFR, AKT, and ERK. Actin was used as a loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 E-cadherin regulates EGFR tissue polarization and activity. a Newborn epidermal whole-mount fluorescence analysis for EGFR and ZO-1 showing enrichment of EGFR at TJs in control SG2 layer. b Quantification of junctional EGFR fluorescence intensity. n = 6 biological replicates with 10 junctions each. Graph shows mean values +- SEM and single means of biological replicates (dots/squares). Granular layer (SG) intensity was normalized to spinous layer (SS) intensity. * P = 0.0214 with D'Agostino & Pearson omnibus normality test followed by one sample t -test. c Newborn epidermal whole-mount fluorescence analysis for EGFR and ZO-1 showing SG2, SS, and cross section (v. section), showing increased junctional ZO-1 and EGFR (arrowheads) in the SS of E-cadherin (Ecad) -/- epidermis compared to control (Ctr). a , c Partial projections and v. sections of confocal stacks from newborn epidermal whole mounts. For better visualization fluorescence intensities of SS projections have been increased relative to SG. Nuclei were stained with DAPI (blue). d Quantification of cortical EGFR enrichment in SG normalized to Ctr SS. Shown are respective means (lines) of three biological replicates with 20 measurements (dots) per replicate, * P < 0.05; n = 3 biological replicates with Kruskal-Wallis, Dunn's post hoc test. e Western blot for total and phosphorylated EGFR (pEGFR) from Ctr and Ecad epi-/- newborn epidermis. f Western blot quantification of pEGFR levels in newborn Ctr and Ecad epi-/-

Supportive validation

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Experimental details: 3 Fig. EWI-2 removal promotes tumor cell motility by regulating the enrichment of exosomal miR-3934-5p. (A) Exosomes from PC3 NEG and KO cells were purified and miRNA of exosomes was extracted. Real-time PCR for miRNA was carried out and the miR-29a, miR-149-3p and miR-3934-5p expression levels in exosomes were detected. The relative miRNA expression level was quantified with Student's t -test and presented as mean +- SD ( n = 3 individual experiments). * P < 0.05; ** P < 0.01. (B) PC3 NEG and KO cells were treated with miR-29a, miR-149-3p and miR-3934-5p as well as miRctrl mimics and analyzed by western blot assay. The miR-3934-5p mimic significantly inhibited the EGFR signaling in PC3 cells with downregulated p1068-EGFR and the phosphorylated p44/42-MAPK (ERK1/2). (C) The normalized band densities of (B) were quantified with Student's t -test and are presented as mean +- SD ( n = 3 individual experiments). * P < 0.05; ** P < 0.01; *** P < 0.001. (D) The predicated interaction between miR-3934-5p and the EGFR 3'-untranslated region (3'-UTR) in TargetScan database. (E) PC3 NEG and KO cells were treated with EGFR inhibitor gefitinib and miR-3934-5p mimic, as well as DMSO as negative control and then examined for the migration through Transwell inserts, which were coated with either fibronectin (10 mug*mL -1 ) or laminin 111 (10 mug*mL -1 ). The cells that migrated through the insert pores and adhered to the bottom of the inserts were photographed. Scale bars: 200 um. (F) Numbe

Supportive validation

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Experimental details: Figure 4 The EGFR-Lpd interplay is cell-line-dependent. ( A ) Representative immunoblot of EGFR, Lpd, phosphorylated Lpd (Y426) and beta-actin in A172 and U343MG upon EGFR knockdown and 1 h after irradiation. ( B ) Densitometric analyses showing changes in Lpd expression and phosphorylation from ( A ). Fold changes of Lpd levels are calculated by normalization to beta-actin. Fold changes of phosphorylated Lpd Y426 are determined by normalization to total Lpd expression. All protein levels were related to the corresponding beta-actin. Fold changes are relative to control-treated and unirradiated samples. ( C ) Representative Western blot from Lpd-silenced A172 and U343MG showing total and phosphorylated EGFR 1 h post-X-ray irradiation. beta-actin served as loading control. Representative Western blots are shown. ( D ) Densitometry of EGFR from ( C ). Fold changes of EGFR levels are calculated by normalization to beta-actin. Fold changes of phosphorylated EGFR are calculated by normalization to total EGFR expression. All protein levels were related to the corresponding beta-actin. Fold changes are relative to control-treated and unirradiated samples. All results are mean +- SD ( n = 3, two-sided Student's t -test, * p < 0.05, ** p < 0.01, *** p < 0.001). si, small interfering RNA; C, control; Lpd, Lamellipodin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Sensitivity of lung cancer cell lines to EGFR-TKIs A. MTS assay for PC-9, H3255, PC9-ER, and H1975 cells. The IC50 values (nM) for EGFR-TKIs are shown. Error bars indicate standard deviation. B. The results of immunoblotting for PC-9, H3255, PC9-ER, and H1975 cells are shown. The cells were treated with the indicated concentrations of EGFR-TKIs for 4 h. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. pEGFR, pAKT, and pERK indicate the phosphorylated form of EGFR, AKT, and ERK, respectively. Actin was used as a loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Efficacy of EGFR-TKIs for EGFR exon 20 insertion mutations A. MTS assay (left) and immunoblotting (right) for BID007 (EGFR A763_Y764insFQEA) cells. Error bars indicate standard deviation. B. Results of immunoblotting for Ba/F3 cells with EGFR exon 20 insertion mutations. The cells were treated with the indicated concentrations of EGFR-TKIs for 4 h. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. pEGFR, pAKT, and pERK indicate the phosphorylated form of EGFR, AKT, and ERK, respectively. Actin was used as a loading control. C. Apoptosis assay using cytometry. Ba/F3 cells harboring wild type EGFR and EGFR D770_N771insNPG (NPG) were treated with EGFR-TKIs for 48 h, subsequently the cells were stained with propidium iodide and annexin V-APC. The numbers indicate the proportion of annexin V-positive and/or propidium iodide-positive cells.

Supportive validation

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Experimental details: Figure 1 Kinetic and concentration-dependent effects of trastuzumab and afatinib on the activation of EGFR and HER 2 in NCI -N87 cells. The levels of activated receptors were determined after treatment of NCI -N87 cells with trastuzumab and/or afatinib by western blot analysis. Activation of the receptors was analyzed using pEGFR -specific (Y1068) and pHER 2-specific (Y1221/2) antibodies. (A) The levels of activated receptors were determined after 5 min of treatment of NCI -N87 cells with afatinib (0.01, 0.1, 0.5, 1 mu m ) and/or trastuzumab (5 mug*mL -1 ). (B) Cells were treated for 5, 20, 60, and 120 min with trastuzumab (5 mug*mL -1 ) and/or afatinib (0.5 mu m ). Equal loading of the lanes was confirmed by the detection of alpha-tubulin or beta-actin. The depicted results are representative of three independent experiments. The results of the densitometric analysis and P -values are shown in the Supplement (Fig. S2 , Fig. S3 , Table S1 , and Table S2 ). A, afatinib; T, trastuzumab; Ctrl, control.