44-794G

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Provider product page for 44-794G

Western blot

ELISA

Antibody data

Antibody Data
Antigen structure
References [23]
Comments [0]
Validations
Immunocytochemistry [1]
Immunohistochemistry [2]
Flow cytometry [1]
Other assay [13]

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Product number: 44-794G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-EGFR (Tyr1173) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

EGFR protein structure - 44-794G shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of EGFR (pY1173) showing staining in the membrane and cytoplasm 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 (pY1173) Rabbit Polyclonal Antibody (Product # 44-794G) diluted in 3% BSA-PBS at a dilution of 1:50 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 (pY1173) 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 (pY1173) Rabbit Polyclonal Antibody (Product # 44-794G) 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: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Peptide ELISA showing of Phospho-EGFR (Tyr1173) using a polyclonal antibody (Product # 44-794G).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 EGFR and ERK1/2 phosphorylation following treatment of SCC61 cells with test compounds. a Averaged quantification of Western blots analyzing Y 1068 - or Y 1173 -phosphorylated EGFR following 1 h pre-treatment with compounds, vehicle, cetuximab, or erlotinib, followed by 45 min treatment with vehicle or EGF +/- EGF. Values shown are normalized to total EGFR. b Averaged quantification of Western blots analyzing T 202 /Y 204 -phosphorylated ERK1/2 following 1 h pre-treatment with compounds, vehicle, cetuximab, or erlotinib, followed by 45 min treatment with vehicle or EGF +/- EGF. Values shown are normalized to total ERK1/2. c Representative Western blots for quantified data shown in parts a and b

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Constitutive activation of EGFR sustains cell proliferation in the absence of ligands a. Serum starved PC3, DU145, A549 and HT-29 cells were tested for EGFR phosphorylation. b. PC3 and A549 cells grown in the absence of serum/ligands were tested for EGFR dimerization (crosslinked by DMP) and phosphorylation. Serum starved A549 and PC3 were treated with EGF +/- C225 at 2.5 ug/ml c. or AEE788 at 2.5 uM d. for 15 minutes and measured for EGFR phosphorylation, EGFR and Actin using Western blot. e. Schematic diagram of WT and extra cellular domain deleted EGFR (DeltaECD-EGFR). f. Western blot analysis of protein samples for pEGFR and EGFR (Flag) isolated from HEK 293 cells transfected with vector alone, WT EGFR or DeltaECD EGFR for 24 hours. g. Western blot analysis of protein samples for pAkt, Akt, pErk1/2, Erk1/2 and EGFR isolated from PC3 cells treated with EGF +/- AEE788 or EGF +/- C225 for 15 minutes similarly as cells used in c and d. h. A549 cells were treated with AEE788 or C225 for 5 days at the indicated concentrations and measured cell number. i. Colony formation assay on PC3 and A549 cells were treated with increasing concentrations of AEE788 or C225 as indicated in 6-well plate and colony formation was counted when the cells reached 80-90% confluence. Data are means of +/- SD of triplicates ( Suppl Figure 1a and 1b ). Asterisk indicates the statistical significance between treated group and DMSO ( P -value

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Activation of EGFR by de novo fatty acid synthesis dependent palmitoylation a. Western blot analysis of protein samples for pEGFR and EGFR isolated from PC3 cells treated with FASN inhibitor, cerulenin, at 5ug/ml for 8 hours. b. PC3 cells were treated with cerulenin for 8 hours and isolated protein samples were cross linked with BS3 as described in the materials and methods section. The cross linked samples were analyzed using Western blot for EGFR dimers. For control group, protein samples were left untreated. c. HEK 293 cells were transfected with DeltaECD EGFR for 24 hours followed by treatment with cerulenin in serum free medium for overnight before protein isolation and Western blot analysis. d. Western blot analysis of protein samples for pEGFR, EGFR and Actin isolated from MCF10A cells transfected with increasing FASN (Flag tagged) concentration for 24 hours followed by serum starvation for 12 hours. e. Western blot analysis of protein samples (for indicated proteins) isolated from PC3 cells transfected with vector alone or FASN flag for 24 hours followed by serum starvation for 12 hours. f. Acyl-Biotin Exchange assay (ABE) for EGFR palmitoylation. PC3 cells were transfected with vector alone or FASN (panel 2e) and isolated samples were subjected to ABE assay for EGFR palmitoylation. HAM stands for hydroxyl amine. ABE assay was performed as described in the methods. g. Western blotting analysis of protein samples for pEGFR, EGFR and actin isolated from PC3 cel

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 PAT 1, 2 and 21 increases EGFR activation and palmitoylation a. Screening of PAT enzymes for EGFR activation. A549 cells were transfected with individual PAT (HA-tagged) constructs for 24 hours followed by 12 hours of serum starvation. Isolated protein samples were tested for EGFR activation using pEGFR antibody. b. Western blot analysis of protein samples for pEGFR, EGFR, pAkt, Akt, pErk 1/2, Erk 1/2 and Actin isolated from A549 cells transfected with PAT plasmids 1, 2, 21 or vector alone for 24 hours followed by serum starvation for 12 hours. c. Western blot analysis of immunoprecipitated samples for HA and EGFR antibodies. HEK 293 cells were transfected with EGFR-flag alone or in combination with indicated PATs as shown in the Figure 3c and PAT enzymes were immunoprecipitated with HA antibody. d. Western blotting analysis of palmitoylated EGFR from PC3 cells transfected with vector alone or PATs.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 C797 site is conserved in kinase active ErbB members and is important for constitutive activation and palmitoylation of EGFR a. Comparison of palmitoylation site (C797) of EGFR with HER2, HER3 and HER4. b. Western blotting analysis for palmitoylated EGFR prepared from HEK293 cells transfected with EGFR WT or C797G mutant (Flag-tagged). c. C797G mutation blocks EGFR constitutive phosphorylation when expressed in HEK 293 cells. d. C797G mutation blocks palmitoylation of DeltaECD EGFR in HEK 293 cells. e. C797G mutation blocks DeltaECD EGFR constitutive phosphorylation in HEK 293 cells. f. HEK 293 cells were transfected with WT or C797G EGFR for 24 hours and treated with DMSO or EGF at 10 ng/ml for 20 minutes and protein samples were analyzed for EGFR dimers using Western blotting under reducing or non-reducing conditions. g. The same samples from panel f were subjected to Western blot analysis. h. Western blot analysis for Flag, pErk 1/2, Erk 1/2 and GAPDH. Protein samples were prepared from HEK 293 cells transfected with WT or cysteine mutants of ErbB members.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 FASN or PAT inhibitor sensitizes cancer cells to EGFR TKIs Western blot analysis of protein samples for pEGFR, EGFR and Actin isolated from A549 cells treated (30 minutes) with increasing concentrations of EGFR tyrosine kinase inhibitor in the presence or absence of cerulenin at 5 ug/ml a. or 2-BP at 8 uM b. The cells were pretreated with either cerulenin or 2-BP for 5 hours before the treatment with Iressa. Western blot analysis of protein samples for pEGFR, EGFR and Actin isolated from PC3 cells treated (30 minutes) with increasing concentrations of EGFR tyrosine kinase inhibitor in the presence or absence of cerulenin at 5 ug/ml c. or 2-BP at 8 uM d, e. MTS assay in PC3 cells treated with EGFR TKI alone or in combination with cerulenin or 2-BP for 48 hours. f. MTS assay in A549 cells treated with EGFR TKI alone or in combination with cerulenin or 2-BP for 48 hours. Data are means of +/- SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO ( P -value

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Drug inhibition of targets in Pkd1 -/- and wt mice . (A) Quantification of Western data for indicated proteins from kidney lysates prepared from vehicle-treated wt or Pkd1 -/- mice, normalized to beta-actin or vinculin-loading control. * P < 0.05 and ** P < 0.01 relative to vehicle treated. (B) Aurora-A (AURKA) was immunoprecipitated from kidney lysates and used for in vitro kinase with gamma-32P-ATP to indicate autophosphorylation and phosphorylation of histone H3 kinase (HH3) (top two rows), with parallel blots probed by Western to allow normalization to total AURKA and HH3 in reaction. Quantitation of data from complete cohort of mice in each treatment group, indicating ratio of phosphorylated HH3 or AURKA to total immunoprecipitated AURKA in wt and Pkd1 -/- mice following indicated drug treatments. * P < 0.05, ** P < 0.01, and *** P < 0.001 relative to vehicle treated. (C) Western analysis showing representative expression of total Aurora-A (tAURKA) in kidney lysates after 10 weeks of treatment with indicated drugs, with beta-actin loading control. (D) Quantitation of data from complete cohort of mice in each treatment group, indicating ratio of total AURKA to beta-actin in wt and Pkd1 -/- mice following indicated drug treatments. ** P < 0.01 relative to vehicle treated. (E) Western blot with representative images of kidney lysates from each treatment cohort showing expression of EGFRphosphorylated (ph) at the indicated amino acids, total EGFR (tEGFR) or beta-act

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Downmodulation of EGFR and c-Met in H1975-HGF xenograft tumors upon JNJ-61186372 treatment. Female nude mice bearing H1975-HGF lung cancer xenografts were treated with 10 mg/kg of the indicated antibodies or control on study days 1 and 4. Tumors were collected 24 hr after the second dose (day 4). Levels of total EGFR (A), c-Met (C) and ERK1 (p44) (E) and phosphorylated c-Met (pc-MET)(B), pEGFR (D), and pERK1 (p44) (F) were analyzed by Western blot. Graphs show densitometry analysis normalized to actin. # (p < 0.05) indicates statistical significant difference compared to vehicle control (Sidak's multiple comparisons test on normalized signal values).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Comparison between nor UDCA- and TUDC-induced Erk-1/-2, p38 MAPK and EGFR activation. Rat livers were perfused with nor UDCA or TUDC (20 umol/l each) for up to 60 min as described in ""Experimental Procedures"". Liver samples were taken at the time points indicated. Phosphorylation of Erk-1/-2, p38 MAPK , and EGFR tyrosine residues Tyr 845 , Tyr 1045 , and Tyr 1173 was analyzed by ( a ) Western blot using specific antibodies and ( b ) subsequent densitometric analysis (black squares, nor UDCA; gray squares, TUDC). Total Erk-1/-2, total p38 MAPK , and total EGFR served as respective loading controls. Phosphorylation at t = 0 was arbitrarily set to 1. Data represent the mean (mean +- SEM) of at least three independent experiments; * p < 0.05 statistical significance compared with the unstimulated control. # p < 0.05 statistical significance between nor UDCA and TUDC. Blots were cropped to focus on the area of interest, and full-length blots are presented in Supplementary Figure 10 . TUDC led to activation of Erk-1/-2, p38 MAPK , and EGFR, as indicated by phosphorylation of the EGFR tyrosine residues Tyr 845 and Tyr 1173 . nor UDCA induced a transient Erk-1/-2 phosphorylation and a weak p38 MAPK activation. No EGFR activation was observed in nor UDCA-perfused livers.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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: 10.1371/journal.pone.0231711.g007 Fig 7 Reciprocal expression of AnxA6 and GRF2 in TNBC PDX models. A) Whole tissue extracts were prepared from patient derived xenografts (n = 19), and equal amounts of protein were analyzed by western blotting using antibodies to the indicated proteins. B) Densitometric analysis of the expression of the indicated proteins. Shown are protein band intensities normalized to beta-actin from a representative experiment. C) The ratio of GRF2:AnxA6 was used to classify the tumors into potentially rapidly growing or invasive tumors as in Fig 6 . D) A plot of the reciprocal expression of AnxA6 and GRF2 based on the GRF2:AnxA6 ratio for each PDX sample and showing two major groups: Group 1 with a strong potential for rapid growth and Group 3 with a strong potential for invasiveness, separated by a more diverse Group 2 with varied potentials for growth and invasiveness.