egfr  (Santa Cruz Biotechnology)

Journal: Journal of Cancer

Article Title: EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells

doi: 10.7150/jca.44234

Figure Lengend Snippet: IRE1α-XBP1s pathway and EGFR expression in colorectal cancer. A. Immunoblotting of EGFR, IRE1α and XBP1s in paired tumor and normal tissues of colorectal cancer patients. GAPDH was used as a loading control. B. Expression of spliced XBP1 mRNA in 6 paired patient samples via q-PCR (n=6). Values are represented as the mean ± SD (n = 3) for each treatment (*P<0.05.) C. Correlation of EGFR and XBP1 in colorectal cancer patients (GSE 38832). D. Immunoblotting of EGFR, IRE1α and XBP1s in colorectal cancer cell lines. E. Expression of spliced XBP1 mRNA in colorectal cancer cell lines via q-PCR. Values are represented as the mean ± SD (n = 3) for each treatment (***P<0.001, ****P<0.0001.).

Article Snippet: Primary antibodies were showed as follow: IRE1α (Cell Signaling Technology, 3294S); p-IRE1α (abcam, ab124945); EGFR (Cell Signaling Technology, 4277S); p-EGFR (Cell Signaling Technology, 3777S); ERK1/2 (Cell Signaling Technology, 9102S); p-ERK1/2 (Cell Signaling Technology, 9101S); XBP1 (Invitrogen, catalog number: PA5-27650); GAPDH (60004-1-Ig); Anti-mouse or anti-rabbit IgG HRP-linked secondary antibodies was purchased from OriGene.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Cancer

Article Title: EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells

doi: 10.7150/jca.44234

Figure Lengend Snippet: EGFR activation is associated with IRE1α -XBP1s Signaling. A. Phosphorylation of EGFR and IRE1α were examined in SW480 cells 30min after stimulation of EGF (50ng/ml). B. Expression of spliced XBP1 mRNA in SW480 cells30 min after stimulation of EGF (50ng/ml). Values are represented as the mean ± SD (n = 3) for each treatment (*P<0.05.) C. Phosphorylation of EGFR and IRE1α were examined in HCT116 cell line 30min after stimulation of EGF (50ng/ml) via immunoblotting. D, E. Activation of IRE1α-XBP1s pathway in SW480 and HCT116 were examined via immunoblotting.SW480 cells (D) and HCT116 (E) cells were transfected with pcDNA6.0-EGFR and incubated for 36h before harvest. F. Activation of IRE1α-XBP1s pathway was examined in SW480 cells 36h after transfection with siRNA oligos. G. Activation of IRE1α-XBP1s pathway was examined in HCT116 and HCT116 EGFR KO cells. H, I. Phosphorylation of EGFR, IRE1α and spliced XBP1 were detected in SW480 (H) and HCT116 (I) treated with cetuximab(12.5μg/ml) and Gefitinib(10nM)for 24hrs. J. mRNA of spliced XBP1 was detected in SW480 24 hrs after cetuximab (12.5μg/ml) treatment. Values are represented as the mean ± SD (n = 3) for each treatment (**P<0.01.) K. Immunocytochemistry assay was conducted to detect proteins of p-EGFR and p-IRE1α. HCT116 cells were cultured on 14mm coverslips and treated with cetuximab(12.5μg/ml) for 24hrs. L. Expression of spliced XBP1 mRNA was assessed in SW480 cells with different treatment. Cells were transfected with pcDNA6.0-EGFR and then treated with cetuximab(12.5μg/ml) for 24hrs before harvest. Values are represented as the mean ± SD (n = 3) for each treatment (*P<0.05, ***P<0.001) M. Expression of spliced XBP1 mRNA was assessed in SW480 cells, which were transfected with pcDNA3.0-HA-IRE1α and then treated with cetuximab (12.5μg/ml) for 24hrs before harvest. Values are represented as the mean ± SD (n = 3) for each treatment (***P<0.001, ****P<0.0001.)

Article Snippet: Primary antibodies were showed as follow: IRE1α (Cell Signaling Technology, 3294S); p-IRE1α (abcam, ab124945); EGFR (Cell Signaling Technology, 4277S); p-EGFR (Cell Signaling Technology, 3777S); ERK1/2 (Cell Signaling Technology, 9102S); p-ERK1/2 (Cell Signaling Technology, 9101S); XBP1 (Invitrogen, catalog number: PA5-27650); GAPDH (60004-1-Ig); Anti-mouse or anti-rabbit IgG HRP-linked secondary antibodies was purchased from OriGene.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Western Blot, Transfection, Incubation, Immunocytochemistry, Cell Culture

Journal: Journal of Cancer

Article Title: EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells

doi: 10.7150/jca.44234

Figure Lengend Snippet: EGFR signaling activates IRE1α through the kinase activity of ERK. A. The molecules of EGFR pathway were detected in SW480 cells by immunoblotting. B. The molecules of EGFR pathway of HCT116 EGFRKO cell line and HCT116 cells were examined by immunoblotting. C. EGFR downstream pathway were detected by immunoblotting in HCT116 treated with cetuximab (12.5μg/ml) for 24hrs. D. Proteins of IRE1α-XBP1s pathway in HCT116 cells with the treatment of MEK inhibitor PD0325901 (1nM) for 24hrs were assessed by immunoblotting. E. p-IRE1α (S724) and XBP1s were assessed via immunoblotting after the treatment of MEK inhibitor PD0325901 (1nM) or ERK inhibitor SCH8477 (5μM) for 24hrs. F. Expression of spliced XBP1 mRNA was assessed in SW480 cells after treatment with MEK inhibitor PD0325901 (1nM) or ERK inhibitor SCH8477 (5μM). MKC8866 was used as positive control. Values are represented as the mean ± SD (n = 3) for each treatment (****P<0.0001.) G, H. Expression of spliced XBP1 mRNA was assessed in SW480 (G) and HCT116 (H) cells after treatment with ERK inhibitor Ravoxertinib(10nM)for 24hrs. Values are represented as the mean ± SD (n = 3) for each treatment (****P<0.0001.) I, J. Coimmunoprecipitation was conducted in SW480 cell line.

Article Snippet: Primary antibodies were showed as follow: IRE1α (Cell Signaling Technology, 3294S); p-IRE1α (abcam, ab124945); EGFR (Cell Signaling Technology, 4277S); p-EGFR (Cell Signaling Technology, 3777S); ERK1/2 (Cell Signaling Technology, 9102S); p-ERK1/2 (Cell Signaling Technology, 9101S); XBP1 (Invitrogen, catalog number: PA5-27650); GAPDH (60004-1-Ig); Anti-mouse or anti-rabbit IgG HRP-linked secondary antibodies was purchased from OriGene.

Techniques: Activity Assay, Western Blot, Expressing, Positive Control

Journal: The American Journal of Pathology

Article Title: Combined Systemic Disruption of MET and Epidermal Growth Factor Receptor Signaling Causes Liver Failure in Normal Mice

doi: 10.1016/j.ajpath.2018.06.009

Figure Lengend Snippet: EGFR, MET, and AKT signaling pathways in EGFRi, ΔMET + EGFRi, and ΔMET mice. A: Western immunoblot analysis of key signaling proteins and their phosphorylated (activated) derivatives downstream of MET and EGFR signaling pathways. B: The phosphatidylinositol 3-kinase (PI3K)/AKT effector pathway. Ponceau staining was used as a loading control; regions shown are at the molecular weight region of the protein shown in the Western blot immediately above. mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; TCPTP, T-cell protein tyrosine phosphatase.

Article Snippet: Antibodies obtained from Cell Signaling Technology (Danvers, MA) are the following: EGFR retinoblastoma (Rb) Ab (number 2640); phosphorylated (phospho)-EGFR (Y1068) Rb Ab (number 3777); phospho-MET (Y1349) Rb Ab (number 3121); MET mouse Ab (number 4560) (although it had not been tested on mice, it worked well in our samples); PI3K p110α Rb Ab (number 4249); PI3K p85 Rb Ab (number 4292); phospho-AKT (S473) Rb Ab (number 9271); phospho-AKT (T308) Rb Ab (number 9275); mammalian target of rapamycin (mTOR) Rb Ab (number 2983); phospho-mTOR (S2448) Rb Ab (number 5536); phospho-mTOR (S2481) Rb Ab (number 2974); non–phospho-phosphatase and tensin homolog (PTEN) Rb Ab (number 7960); PTEN Rb Ab (number 9188); cyclin D1 Rb Ab (number 2922); STAT3 Rb Ab (number 4904); phospho-STAT3 (Tyr705) Rb Ab (number 9131); phospho–glycogen synthetase kinase (GSK)-3β (Ser9) Rb Ab (number 9323); phospho-p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) (T202/Y204) Rb Ab (number 9101); p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) mouse Ab (number 4696); phospho-AMPKα (Thr172) Rb Ab (number 2535); AMPKα Rb Ab (number 5831); T-cell protein tyrosine phosphatase (TC45) Rb Ab (number 58935); and Apoptosis Antibody Sampler Kit (Mouse Preferred; number 9930).

Techniques: Protein-Protein interactions, Western Blot, Staining, Control, Molecular Weight

Journal: Oncotarget

Article Title: GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity

doi: 10.18632/oncotarget.12600

Figure Lengend Snippet: A. Western blot analysis of EGFR activation, as demonstrated by phosphorylation at Y1173, in control (Ctrl) and erlotinib (Erl) treated pEGFR Hi tumor cells. B. EGFR inhibition increases adhesion of pEGFR Hi cells to a collagen IV-coated substrate. C. Phosphorylation of EGFR at Y1173 in pEGFR Lo cells treated with 0, 50 μM, and 500 μM of the phosphatase inhibitor sodium orthovanadate (NaOV). D. Reduced adhesion of pEGFR Lo cells to collagen IV in the presence of increasing concentrations of NaOV. E. NaOV-induced reduction in adhesion to collagen IV is restored by inhibition of EGFR by Erl in pEGFR Lo cells. H - J. Representative confocal images of phalloidin labeled f-actin (green) and nuclei (blue) in control (H), NaOV (I), and NaOV+erlotinib (J) treated pEGFR Lo cells 2 hours after plating. p<0.05 = *, p<0.01 = **. Error bars are displayed as standard error of mean (SEM). (B) n=6, (D) n=3, (E) n=8. Scale bars F-H = 20 μm. (B, D, E) Representative data from triplicate experiments.

Article Snippet: Rb α Phospho EGFR Y1173 , Cell Signaling , 53A5 , 4407 , WB , 1:500.

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Control, Inhibition, Labeling

Journal: Oncotarget

Article Title: GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity

doi: 10.18632/oncotarget.12600

Figure Lengend Snippet: Antibodies used

Article Snippet: Rb α Phospho EGFR Y1173 , Cell Signaling , 53A5 , 4407 , WB , 1:500.

Techniques: Concentration Assay, Immunohistochemistry-IF