Journal: Oncogene

Article Title: Tamoxifen down-regulation of miR-451 increases 14-3-3ζ and promotes breast cancer cell survival and endocrine resistance

doi: 10.1038/onc.2011.223

Figure Lengend Snippet: Immunoblotting for pHer2, pEGFR, pAKT, pMAPK, and 14-3-3ζ in Tam R cells treated with heregulin (HRG) for the minutes indicated after cells were transfected with A) control or 14-3-3ζ expressing polylysine-coated adenovirus or B) control or pre-miR-451 vector to overexpress miR-451. β-actin was used as a loading control.

Article Snippet: Western blotting used antibodies against 14-3-3ζ (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (Sigma-Aldrich Corp., St. Louis, MO), pEGFR, pHER2, pMAPK and pAKT (Cell Signaling).

Techniques: Western Blot, Transfection, Control, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

doi: 10.1371/journal.pone.0055657

Figure Lengend Snippet: Fourteen EGFR tyrosine, serine and threonine phosphorylation sites were screened via in situ PLA and immunoblotting analyses.

Article Snippet: The monoclonal antibodies used included those targeting pEGFR-Tyr1068 (Cell Signaling), EGFR (Cell Signaling), pAKT-Ser473 (Cell Signaling), pAURKA-Thr288 (Abcam), EGFR (Abcam), AKT (Cell Signaling) and AURKA (BD Bioscience).

Techniques: In Situ, Western Blot

Journal: PLoS ONE

Article Title: Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

doi: 10.1371/journal.pone.0055657

Figure Lengend Snippet: A431 cells were serum-starved for 16 hours and then treated with EGF (10 ng/ml) in serum-free medium for 10 minutes. (A) In situ PLA is highly sensitive for detection of the phosphorylation of pEGFR-Tyr1068 after EGF stimulation. (B) The quantification of these two signals is shown. (C) EGFR and pEGFR-Tyr1068 were detected in A431 cells by immunoblotting analysis.

Article Snippet: The monoclonal antibodies used included those targeting pEGFR-Tyr1068 (Cell Signaling), EGFR (Cell Signaling), pAKT-Ser473 (Cell Signaling), pAURKA-Thr288 (Abcam), EGFR (Abcam), AKT (Cell Signaling) and AURKA (BD Bioscience).

Techniques: In Situ, Western Blot

Journal: PLoS ONE

Article Title: Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

doi: 10.1371/journal.pone.0055657

Figure Lengend Snippet: H1299 cells stably expressed the empty vector, EGFR-WT or mutant EGFR-L858R. (A) After stimulation with EGF (10 ng/ml) in serum-free medium for 10 minutes, cell extracts were subjected to immunoblotting analysis with the indicated phospho-tyrosine antibodies. EGF-dependent (EGFR-Tyr992, -Tyr1148 and -Tyr1068) and EGF-independent [EGFR-Tyr845, -Tyr974, -Tyr1086 and -Tyr1101, -Thr654 (see below) and -Ser1046 (see below)] phosphorylation were observed in H1299 cells expressing EGFR-L858R mutant. The phosphorylation of EGFR-Thr654 (B) and -Ser1046 (C) with or without EGF treatment in different lung cancer cell lines was monitored via in situ PLA. The quantification of in situ PLA is shown on the right. EGFR-Thr654 and -Ser1046 were phosphorylated upon EGF treatment in H1299-EGFR-WT cells, whereas their phosphorylation was EGF-independent in H1299-EGFR-L858R cells. (D) Immunoblotting analysis of EGFR-Thr654 and -Ser1046 was performed in EGFR mutant cells (H1975, which contains the EGFR-L858R and -T790M mutants) and cells expressing EGFR-WT (A549). The phosphorylation of EGFR-Tyr1068 was induced by EGF. The phosphorylation of EGFR-Thr654 and -Ser1046 was EGF-independent in H1975 and H1299-EGFR-L858R cells as determined by immunoblotting.

Article Snippet: The monoclonal antibodies used included those targeting pEGFR-Tyr1068 (Cell Signaling), EGFR (Cell Signaling), pAKT-Ser473 (Cell Signaling), pAURKA-Thr288 (Abcam), EGFR (Abcam), AKT (Cell Signaling) and AURKA (BD Bioscience).

Techniques: Stable Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, In Situ

Journal: PLoS ONE

Article Title: Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

doi: 10.1371/journal.pone.0055657

Figure Lengend Snippet: (A) The phosphorylation of EGFR-Tyr1068 was faster than that of EGFR-Thr654 and EGFR-Ser1046 under the EGF (10 ng/ml) stimulation. (B) The cells were serum-starved in the presence of VE-465 (1 nM or 100 nM), which is an AURKA inhibitor, for 16 hours and then treated with EGF (10 ng/ml) for 10 minutes. The phosphorylation of EGFR-Thr654 and EGFR-Ser1046, but not pEGFR-Tyr1068, was suppressed by VE-465. (C) The cells were serum-starved in the presence of Iressa (10 µM) for 16 hours and then treated with EGF (10 ng/ml) for 10 minutes. Iressa, which is an EGFR inhibitor, was used to monitor EGFR signaling in H1299 cells stably expressing EGFR-WT and EGFR-L858R mutant. The phosphorylation by AURKA at Thr288 was suppressed by Iressa in both cell lines. (D) H1975 cells were treated with VE-465 and/or Iressa (10 µM) for 16 hrs. VE-465 and Iressa can suppress pEGFR-Thr654 and -Ser1046 in H1975.

Article Snippet: The monoclonal antibodies used included those targeting pEGFR-Tyr1068 (Cell Signaling), EGFR (Cell Signaling), pAKT-Ser473 (Cell Signaling), pAURKA-Thr288 (Abcam), EGFR (Abcam), AKT (Cell Signaling) and AURKA (BD Bioscience).

Techniques: Stable Transfection, Expressing, Mutagenesis

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Type 2 diabetes mellitus facilitates endometrial hyperplasia progression by activating the proliferative function of mucin O-glycosylating enzyme GALNT2.

doi: 10.1016/j.biopha.2020.110764

Figure Lengend Snippet: Fig. 4. GALNT2 disturbance facilitates proliferation and endometrial hyperplasia progression by modifying phosphorylation and activity of EGFR. A: Band diagram and statistical map of EGFR、pEGFR、AKT、pAKT、ERK and pERK protein expressions in rat uterus. B: Band diagram and statistical map of GALNT2 protein expression in Ishikawa cells after transfection and high sugar stimulation (n = 3). C: The proliferation of Ishikawa cells after transfection and high sugar stimulation (n = 3). D: Band diagram and statistical map of pAKT, pERK and pEGFR protein expressions in Ishikawa cells after transfection and high sugar stimulation (n = 3). Data are expressed as Mean ± SEM, n = 6, *P < 0.05, **P < 0.01, compared to the N group; ##P < 0.01, compared to the NH group; %P < 0.05, %%P < 0.01, compared to the MOCK-N group; @@P < 0.01, compared to the si-GALNT2-N group.

Article Snippet: Rabbit polyclonal antibody for pEGFR (3777) was from CST, Inc. (Boston, United States).

Techniques: Phospho-proteomics, Activity Assay, Expressing, Transfection