c-series human egfr phosphorylation antibody array 1 kit  (RayBiotech inc)

Journal: Journal of Personalized Medicine

Article Title: Human Papillomavirus Infection and EGFR Exon 20 Insertions in Sinonasal Inverted Papilloma and Squamous Cell Carcinoma

doi: 10.3390/jpm13040657

Figure Lengend Snippet: EGFR phosphorylation sites in IP-SCC. Eight phosphorylation sites in EGFR were investigated using an EGFR Phosphorylation Antibody Array 1 Assay. ( A ): Description of each well. ( B ): Results for IP-SCC1. ( C ): Results for IP-SCC-2. Of the eight sites, Y845 phosphorylation (F1 and F2 wells) was observed in both cases. Neg, negative control; Pos, positive control. Each antibody is spotted in duplicate vertically.

Article Snippet: A RayBio Human EGFR Phosphorylation Antibody Array 1 Assay (RayBiotech, Inc., Peachtree Corners, GA, USA) was used to screen specific EGFR phosphorylation sites (Y845, Y922, Y1045, Y1068, Y1086, Y1173, and Y1046/47) in two IP-SCC patients with ex20ins.

Techniques: Ab Array, Negative Control, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Targeting the Sheddase Activity of ADAM17 by an Anti-ADAM17 Antibody D1(A12) Inhibits Head and Neck Squamous Cell Carcinoma Cell Proliferation and Motility via Blockage of Bradykinin Induced HERs Transactivation

doi: 10.7150/ijbs.9326

Figure Lengend Snippet: Effect of D1(A12) on the phosphorylation of HER family receptors and downstream signalling molecules under BK stimulation in HNSCC cells. (A) SCC13 cells were pre-incubated with different inhibitors for 2 h then treated with 10 nM BK for 10 min, after which the cell extracts were immunoblotted with the indicated antibodies. (B) SCC9 cells were treated in the same way as SCC13 cells and analysed with indicated antibodies. (C-D) Effect of ADAM17 siRNA and ADAM10 siRNA on the phosphorylation of EGFR and ERK in SCC13 cells. Cells were transfected with 3 different siRNAs and non-targeting siRNA. 72 h later, cells were serum starved for 48 h and then challenged with 10 nM BK for 10 min. After cell lysis, equal amount of proteins were analyzed by immunoblotting with indicated antibodies. (E-F) Effect of D1(A12) on the phosphorylation of HER family receptors in SCC13 cells. (E) Cells were treated as in A and analysed by immunoblotting with indicated antibodies. The same cell extract as 6E were exposed to the RayBio human EGFR phosphorylation antibody array 1 as described in M&M. (F) On each membrane, the left upper corner and right lower corner were spotted with positive control used for signal normalisation. The left lower corner was spotted with pan HER 1, 2 and 3. Five different phosphorylation sites of HER2, HER3 and HER4 were detected in the middle part of the membrane. (G) Normalisation of D1(A12) treated signals to human IgG treated signals. Signal intensity for a particular spot was divided by the average signal intensity of 4 positive controls on the same array membrane. Each phosphorylation site was spotted duplicate.

Article Snippet: To investigate whether BK can transactivate HER2, HER3 and HER4 along with EGFR and whether D1(A12) can prevent this induction in SCC13 cells, the RayBio human EGFR phosphorylation antibody array 1 was used to detect the relative levels of phosphorylation at specific sites of human HER family proteins.

Techniques: Incubation, Transfection, Lysis, Western Blot, Ab Array, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Targeting the Sheddase Activity of ADAM17 by an Anti-ADAM17 Antibody D1(A12) Inhibits Head and Neck Squamous Cell Carcinoma Cell Proliferation and Motility via Blockage of Bradykinin Induced HERs Transactivation

doi: 10.7150/ijbs.9326

Figure Lengend Snippet: Characterization of (p-) EGFR, HER2, HER3, (p-) AKT, (p-) ERK1/2 and ADAM10/17 expression in 5 human HNSCC cell lines and normal human keratinocyte cells (KN). Cell lysates (20 µg) were prepared, resolved by SDS-PAGE, and subjected to immunoblotting analysis with the indicated antibodies. SCC9 cells are yellow fluorescent protein (YFP) labelled. The prefix “p-” and “TT” refer to phosphorylation form and total form of the parentheses. β-actin was used as a loading control.

Article Snippet: Cells were washed once with ice-cold PBS and then extracted with cell lysis buffer provided in the RayBio Human EGFR Phosphorylation Antibody Array kit (RayBiotech).

Techniques: Expressing, SDS Page, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Targeting the Sheddase Activity of ADAM17 by an Anti-ADAM17 Antibody D1(A12) Inhibits Head and Neck Squamous Cell Carcinoma Cell Proliferation and Motility via Blockage of Bradykinin Induced HERs Transactivation

doi: 10.7150/ijbs.9326

Figure Lengend Snippet: Effect of D1(A12) on the phosphorylation of HER family receptors and downstream signalling molecules under BK stimulation in HNSCC cells. (A) SCC13 cells were pre-incubated with different inhibitors for 2 h then treated with 10 nM BK for 10 min, after which the cell extracts were immunoblotted with the indicated antibodies. (B) SCC9 cells were treated in the same way as SCC13 cells and analysed with indicated antibodies. (C-D) Effect of ADAM17 siRNA and ADAM10 siRNA on the phosphorylation of EGFR and ERK in SCC13 cells. Cells were transfected with 3 different siRNAs and non-targeting siRNA. 72 h later, cells were serum starved for 48 h and then challenged with 10 nM BK for 10 min. After cell lysis, equal amount of proteins were analyzed by immunoblotting with indicated antibodies. (E-F) Effect of D1(A12) on the phosphorylation of HER family receptors in SCC13 cells. (E) Cells were treated as in A and analysed by immunoblotting with indicated antibodies. The same cell extract as 6E were exposed to the RayBio human EGFR phosphorylation antibody array 1 as described in M&M. (F) On each membrane, the left upper corner and right lower corner were spotted with positive control used for signal normalisation. The left lower corner was spotted with pan HER 1, 2 and 3. Five different phosphorylation sites of HER2, HER3 and HER4 were detected in the middle part of the membrane. (G) Normalisation of D1(A12) treated signals to human IgG treated signals. Signal intensity for a particular spot was divided by the average signal intensity of 4 positive controls on the same array membrane. Each phosphorylation site was spotted duplicate.

Article Snippet: Cells were washed once with ice-cold PBS and then extracted with cell lysis buffer provided in the RayBio Human EGFR Phosphorylation Antibody Array kit (RayBiotech).

Techniques: Incubation, Transfection, Lysis, Western Blot, Ab Array, Positive Control

Journal: Molecular Cancer

Article Title: Targeting EGFR with photodynamic therapy in combination with Erbitux enhances in vivo bladder tumor response

doi: 10.1186/1476-4598-8-94

Figure Lengend Snippet: Phosphorylation statuses of EGFR sites were determined using antibody arrays . Increased phosphorylation of ErbB2(Thr686), ErbB2(Ser1113) and limited phosphorylation of EGFR(Thy845), ErbB2(Tyr1221/1222), ErbB3(Tyr1289) and ErbB4(Tyr1284) sites was seen in the control group. In the monotherapy groups, ErbB2(Thr686), (Ser113) and ErbB4(Tyr1284) sites were phosphorylated. Inhibition of most of the EGFR phosphorylation sites was observed in combination therapy groups except for ErbB2(Thr686) and (Ser1113). (A: Control, B: PDT, C: Erbitux and D: PDT + Erbitux).

Article Snippet: A human EGFR phosphorylation antibody array (RayBioTech, USA) was used to simultaneously detect phosphorylation of 17 different sites for Human EGFR in cell lysates.

Techniques: Inhibition

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA does not significantly change EGFR phosphorylation and binding and activation by EGF. (A) Neither AcoA nor digoxin (both 100 nM) induce phosphorylation of EGFR family members in A549 cells. EGF (100 ng/ml) was used as a positive control. Phosphorylation of EGFR Tyr845 and ErbB2 Tyr1112 after 60 min treatment are quantified. Analysis was performed by protein array. (B) Representative membranes are shown in (C) Neither AcoA nor digoxin (both at 100 nM) affect EGFR phosphorylation induced by EGF (100 ng/ml) as analyzed by a tyrosine kinase protein array. A549 cells were pretreated for 30 min with AcoA, ouabain (both 100 nM), or erlotinib (10 µM) and stimulated with EGF (100 ng/ml) for 60 min. (D) No synergistic cytotoxicity of AcoA and the EGFR inhibitor cetuximab. A549 lung cancer cells were treated with the EGFR inhibitor cetuximab. After 1 h, AcoA (100 nM) was added and after 24 h cell viability was analyzed by XTT. Data are mean ± SEM, N = 3. (E) AcoA and digoxin do not interfere with EGF-induced cell proliferation. Cell proliferation was quantified based on luminescence in luciferase-expressing A549 cells pretreated with 100 nM of AcoA for either 0, 3, or 24 h and stimulated with 100 ng/ml EGF for additional 48 h ( N = 3). Data are mean ± SEM of N = 3 independent experiments, * p < 0.05, ** p < 0.01.

Article Snippet: To evaluate EGFR phosphorylation at distinct phosphorylation sites we used EGFR phosphorylation antibody arrays (Abcam, Cambridge, United Kingdom).

Techniques: Binding Assay, Activation Assay, Positive Control, Protein Array, Luciferase, Expressing

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA leads to endosomal EGFR arrest and inhibits EGF-induced degradation of EGFR (A) AcoA induces endosomal arrest in EGFR biosensor cells. After 1 h preincubation with monensin (1 µM), AcoA, digoxin, doxorubicin (all 100 nM), or vehicle, cells were stimulated with 100 ng/ml EGF and the formation of green fluorescent vesicles indicating activated and internalized EGFR was monitored microscopically. Representative images are shown. The graphs on the right show the number of fluorescent vesicles/cell at the respective time point. Data are mean ± SEM of N = 3 independent experiments, *** p < 0.001. (B) Quantification of cellular EGFR in A549 cells treated as in (A) using a commercial ELISA In-cell ELISA assay. The amount of EGFR was normalized to cell number assessed by crystal violet staining. Data are mean ± SEM of N = 3 independent experiments, * p < 0.05 vs. control, # p < 0.05 vs. EGF treatment group. (C) At the same conditions, EGFR activation was assessed using ELISA for the Y1173 phosphorylation site of EGFR. EGFR phosphorylation is expressed as the ratio of phosphorylated EGFR to total EGFR. Data are mean ± SEM of N = 3, * p < 0.05 vs. control. (D) Src kinase activation was measured as a downstream target of EGFR. A549 cells treated as in (A) were lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, * p < 0.05 vs. control, ** p < 0.01 vs. control, # p < 0.05 vs. EGF treatment group.

Article Snippet: To evaluate EGFR phosphorylation at distinct phosphorylation sites we used EGFR phosphorylation antibody arrays (Abcam, Cambridge, United Kingdom).

Techniques: Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Staining, Activation Assay

Journal: Cancer Gene Therapy

Article Title: B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation

doi: 10.1038/s41417-020-00246-9

Figure Lengend Snippet: Effect of NHL-a101 and NHL-a102 on expression and phosphorylation levels of intracellular signaling proteins.

Article Snippet: To verify vp17-triggered EGR activation, we performed experiments using the RayBio human EGFR phosphorylation antibody array, which simultaneously detects 17 different specific phosphorylation sites on EGFR family members.

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation

doi: 10.1038/s41417-020-00246-9

Figure Lengend Snippet: Cellular extracts of Raji B cells treated for 5 min at 37 °C with or without EGF, NHL-a101 and NHL-a102 were evaluated for phosphorylation of EGFR family members by a human phosphorylation array. The intensities of the phospho-protein signals were quantified by densitometric analysis and normalized to either positive controls or not treated cells as suggested by the manufacturer’s instructions. Values reported for protein phosphorylation levels are representative of one representative experiment of 3 with similar results. NT, not treated.

Article Snippet: To verify vp17-triggered EGR activation, we performed experiments using the RayBio human EGFR phosphorylation antibody array, which simultaneously detects 17 different specific phosphorylation sites on EGFR family members.

Techniques:

Journal: Cancer Gene Therapy

Article Title: B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation

doi: 10.1038/s41417-020-00246-9

Figure Lengend Snippet: Effect of NHL-a101 and NHL-a102 on expression and phosphorylation levels of intracellular signaling proteins.

Article Snippet: The human EGFR phosphorylation antibody array was obtained from RayBiotech (Peachtree Corners, GA, USA) and performed according to manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation

doi: 10.1038/s41417-020-00246-9

Figure Lengend Snippet: Cellular extracts of Raji B cells treated for 5 min at 37 °C with or without EGF, NHL-a101 and NHL-a102 were evaluated for phosphorylation of EGFR family members by a human phosphorylation array. The intensities of the phospho-protein signals were quantified by densitometric analysis and normalized to either positive controls or not treated cells as suggested by the manufacturer’s instructions. Values reported for protein phosphorylation levels are representative of one representative experiment of 3 with similar results. NT, not treated.

Article Snippet: The human EGFR phosphorylation antibody array was obtained from RayBiotech (Peachtree Corners, GA, USA) and performed according to manufacturer’s instructions.

Techniques: