Human Phospho-EGFR (Y1173) Antibody

EGFR in Human Lung Cancer.

EGFR was detected in immersion fixed frozen sections of human lung cancer using Rabbit Anti-Human Phospho-EGFR (Y1173) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1095) at 0.5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (VC003). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cell membrane in cancer cells. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Phospho-EGFR (Y1173) in A431 Human Cell Line by Flow Cytometry.

A431 human epithelial carcinoma cells were untreated (open histogram), or treated for 5 minutes with 100 ng/mL Recombinant Human EGF (Catalog # 236-EG; filled histogram) then stained with Rabbit Anti-Human Phospho-EGFR (Y1173) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1095), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with para-formaldehyde and permeabilized with saponin.

Detection of Human Phospho-EGFR (Y1173) by Simple Western^TM.

Simple Western lane view shows lysates of A431 human epithelial carcinoma cell line untreated (-) or treated (+) with 10 ng/mL Recombinant Human EGF (Catalog # 236-EG) for 5 minutes, loaded at 0.2 mg/mL. A specific band was detected for Phospho-EGFR (Y1173) at approximately 265 kDa (as indicated) using 2 µg/mL of Rabbit Anti-Human Phospho-EGFR (Y1173) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1095). This experiment was conducted under reducing conditions and using the 66-440 kDa separation system.

Detection of Phospho-EGFR (Y1173) by Western Blot

Matrix Fibulin-1 regulates EGF dependent EGFR activation in Calu-1. (A) Endogenous Fibulin-1 (WB: FBLN1) & GAPDH (WB: GAPDH) detected by WB in cell derived matrix (CDM before replating) made from control (CON), FBLN1C (1Ci) & FBLN1D (1Di) knockdown Calu-1, grown with serum (5% FBS) for 72 h. Calu-1 replated on this CDM & grown for 18 h with serum [+18 h (5% FBS)] & levels of Fibulin-1 (WB: FBLN1) & GAPDH (WB: GAPDH) detected by WB in cell derived matrix (CDM after replating) & compared to those seen in CDM before replating. FBLN1C & FBLN1D isoforms detected in blots of knockdown lysates are marked by arrows. Blot is best representative of three independent experiments that gave similar results. (B) Representative individual migration tracks of Calu-1 adherent in the presence of serum growth factors (5% FBS) on CDM (made as detailed above) from control (CON-CDM), FBLN1C (1Ci-CDM), & FBLN1D (1Di-CDM) knockdown Calu-1. (C) Accumulated distance, euclidean distance, velocity & directionality of 100 migrating (per experiment) & represented in the bar graphs as mean ± SE from eight independent experiments. p values calculated using one-way ANOVA & Tukey’s post hoc test & represented if found to be significant. (D,E) WB detection of EGFR phosphorylated on tyrosine 1173 (WB: pEGFR), total EGFR (WB: tEGFR) & GAPDH (WB: GAPDH) in Calu-1 plated on CDM (made as detailed above) from control (CON-CDM), FBLN1C (1Ci-CDM) & FBLN1D (1Di-CDM) knockdown (D) in the presence of serum growth factors (5% FBS) (E) on stimulation with EGF (100 ng/ml) for 5 min (+EGF) in serum deprived Calu-1. Bar graphs represents mean ± SE of pEGFR to total EGFR ratio from 3 or 4 independent experiments as indicated. Statistical analysis of the data was done using the students t-test & p values are as shown. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32719793), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Phospho-EGFR (Y1173) by Western Blot

EGFR inhibition destabilizes HDAC1 protein by promoting its ubiquitination. A PC-9, A549 and H1975 cells were treated with the indicated dose of Gefitinib for 24 h and HDAC1 expression was assessed by Western blot analysis. b Quantification of HDAC1 expression following treatment with the indicated dose of Gefitinib. Graphed values indicate mean ± SEM, n = 4 for PC-9 cells or n = 3 for A549 and H1975 cells. c PC-9, A549 and H1975 cells were treated with 1 µM AG-1478 for 24 h. HDAC1 expression was assessed by Western blot. d PC-9 cells were treated with 1 μM Gefitinib for 24 h and 10 μM MG-132 4 h prior to collection and HDAC1 expression was assessed by Western blot analysis. e HEK293T cells were co-transfected with plasmids expressing HA-tagged ubiquitin and Flag-tagged HDAC1, and were treated with MG-132. Cell lysates were precipitated with an anti-Flag tag antibody and blotted with an anti-HA tag antibody. f HEK293T cells were co-transfected with plasmids expressing His6-tagged ubiquitin and Flag-tagged HDAC1, and were treated with MG-132. Cell lysates were subjected to pulldown with Ni-NTA agarose, and blotted with an antibody to Flag. g Myc-tagged EGFR and either Flag-tagged HDAC1 WT or Y72F mutant were co-expressed in HEK293T cells and were analyzed by Western blot. h HEK293T cells were transfected with plasmids expressing Flag-tagged HDAC1 WT or Y72F mutant. Cells were exposed to 100 µM cycloheximide (CHX) for up to 10 h. At every 2 h, cell lysates were collected and the expression of Flag-tagged HDAC1 was assessed. i Quantification of the expression of Flag-tagged HDAC1. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33976119), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Phospho-EGFR (Y1173) by Western Blot

EGFR inhibition destabilizes HDAC1 protein by promoting its ubiquitination. A PC-9, A549 and H1975 cells were treated with the indicated dose of Gefitinib for 24 h and HDAC1 expression was assessed by Western blot analysis. b Quantification of HDAC1 expression following treatment with the indicated dose of Gefitinib. Graphed values indicate mean ± SEM, n = 4 for PC-9 cells or n = 3 for A549 and H1975 cells. c PC-9, A549 and H1975 cells were treated with 1 µM AG-1478 for 24 h. HDAC1 expression was assessed by Western blot. d PC-9 cells were treated with 1 μM Gefitinib for 24 h and 10 μM MG-132 4 h prior to collection and HDAC1 expression was assessed by Western blot analysis. e HEK293T cells were co-transfected with plasmids expressing HA-tagged ubiquitin and Flag-tagged HDAC1, and were treated with MG-132. Cell lysates were precipitated with an anti-Flag tag antibody and blotted with an anti-HA tag antibody. f HEK293T cells were co-transfected with plasmids expressing His6-tagged ubiquitin and Flag-tagged HDAC1, and were treated with MG-132. Cell lysates were subjected to pulldown with Ni-NTA agarose, and blotted with an antibody to Flag. g Myc-tagged EGFR and either Flag-tagged HDAC1 WT or Y72F mutant were co-expressed in HEK293T cells and were analyzed by Western blot. h HEK293T cells were transfected with plasmids expressing Flag-tagged HDAC1 WT or Y72F mutant. Cells were exposed to 100 µM cycloheximide (CHX) for up to 10 h. At every 2 h, cell lysates were collected and the expression of Flag-tagged HDAC1 was assessed. i Quantification of the expression of Flag-tagged HDAC1. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33976119), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-EGFR (Y1173) by Western Blot

Fibulin-1 is a negative regulator of EGFR activation and function in Calu-1 cells. (G–I) Western blot detection of EGFR phosphorylated on tyrosine1173 (WB: pEGFR), total EGFR (WB: tEGFR), and GAPDH (WB: GAPDH) in lysates from Calu-1 cells (G) in the presence of serum growth factors (5% FBS), (H) on stimulation with EGF (100 ng/ml) for 5 min (+EGF) in serum deprived control (CON), FBLN1C (1Ci), FBLN1D (1Di) knockdown cells and (I) in the presence of serum growth factors (5% FBS) in Calu-1 cells overexpressing untagged FBLN1C or FBLN1D. Overexpression of Fibulin-1C (+FBLN1C) and Fibulin-1D (+FBLN1D) was confirmed by western blot (WB: FBLN1). Arrows mark the position of FBLN1C and FBLN1D isoforms in the representative blots. (G–I) Bar graphs represents mean ± SE of pEGFR to total EGFR ratio from 3 to 5 independent experiments as indicated in each graph. Statistical analysis of the data was done using the students t-test and p values are as shown. (J) Representative images of wound healing assay done in the presence of serum growth factor (5% FBS) in Control (CON) vs. FBLN1C (1Ci) vs. FBLN1D (1Di) knockdown Calu-1 cells at 0 h and 36 h in the presence of DMSO or 10 μM Erlotinib. Images were analyzed using T-Scratch software and the percent closed wound area calculated. Graph represents mean ± SE from four independent experiments. Statistical analysis of the data was done using two-way ANOVA and p values are as shown. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32719793), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-EGFR (Y1173) by Western Blot

Matrix Fibulin-1 regulates EGF dependent EGFR activation in Calu-1. (A) Endogenous Fibulin-1 (WB: FBLN1) & GAPDH (WB: GAPDH) detected by WB in cell derived matrix (CDM before replating) made from control (CON), FBLN1C (1Ci) & FBLN1D (1Di) knockdown Calu-1, grown with serum (5% FBS) for 72 h. Calu-1 replated on this CDM & grown for 18 h with serum [+18 h (5% FBS)] & levels of Fibulin-1 (WB: FBLN1) & GAPDH (WB: GAPDH) detected by WB in cell derived matrix (CDM after replating) & compared to those seen in CDM before replating. FBLN1C & FBLN1D isoforms detected in blots of knockdown lysates are marked by arrows. Blot is best representative of three independent experiments that gave similar results. (B) Representative individual migration tracks of Calu-1 adherent in the presence of serum growth factors (5% FBS) on CDM (made as detailed above) from control (CON-CDM), FBLN1C (1Ci-CDM), & FBLN1D (1Di-CDM) knockdown Calu-1. (C) Accumulated distance, euclidean distance, velocity & directionality of 100 migrating (per experiment) & represented in the bar graphs as mean ± SE from eight independent experiments. p values calculated using one-way ANOVA & Tukey’s post hoc test & represented if found to be significant. (D,E) WB detection of EGFR phosphorylated on tyrosine 1173 (WB: pEGFR), total EGFR (WB: tEGFR) & GAPDH (WB: GAPDH) in Calu-1 plated on CDM (made as detailed above) from control (CON-CDM), FBLN1C (1Ci-CDM) & FBLN1D (1Di-CDM) knockdown (D) in the presence of serum growth factors (5% FBS) (E) on stimulation with EGF (100 ng/ml) for 5 min (+EGF) in serum deprived Calu-1. Bar graphs represents mean ± SE of pEGFR to total EGFR ratio from 3 or 4 independent experiments as indicated. Statistical analysis of the data was done using the students t-test & p values are as shown. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32719793), licensed under a CC-BY license. Not internally tested by R&D Systems.