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: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Phospho-proteomics, 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: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Stable Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Phospho-proteomics, 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) We used ProtoArray™ to identify EGFR as a novel interaction partner for AURKA. Briefly, expressed, recombinant his-tagged human AURKA was probed on ProtoArray™ human protein microarrays at seven concentrations (0, 0.5, 1.0, 5.0, 10, 50 and 100 ng/µl) in duplicate. It should be noted that EGFR-L861Q, but not EGFR-WT, was initially identified to interact with AURKA. (B) HEK293T were co-transfected with Myc-EGFR (EGFR-WT, EGFR-L858R and -L861Q mutants) and Flag-AURKA [AURKA-WT and AURKA-kinase dead (KD)]. After transfection for 48 hours, the cell extracts were subjected to co-immunoprecipitation with an anti-FLAG M2 affinity gel. Protein expression was determined by immunoblotting with anti-Myc and anti-FLAG antibodies. It showed that both EGFR-WT and mutated EGFR associates with AURKA-WT and AURKA-KD. Similar results were observed in at least three independent experiments. (C) The EGFR-AURKA interaction with or without EGF stimulation (10 ng/ml for 10 minutes) was determined via in situ PLA in A549 (EGFR-WT) and H1975 (EGFR-L858R-T790M mutations). Both EGFR-WT and -L858R mutant were associated with AURKA under EGF stimulation. The quantification of in situ PLA signals was shown on the right. (D) The phosphorylation sites of EGFR-Thr654 and -Ser1046 matched the substrate consensus sequences for AURKA. (E) H1975 cells showed more obvious phosphorylation at EGFR-Thr654, EGFR-Ser1046 and AURKA-Thr288 in nocodazole-arrested M-phase than in interphase.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Recombinant, Transfection, Immunoprecipitation, Expressing, Western Blot, In Situ, Mutagenesis, Phospho-proteomics

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: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Mutagenesis

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: Each column indicates a patient and each row indicates an antibody. The IHC intensity in the patients was defined as 0, 1 and 2 for AURKA, pEGFR-Thr654 and pEGFR-Ser1046 staining.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Staining

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: Wilcoxon two-sample t-test for IHC staining in 25 NSCLC patients.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Immunohistochemistry

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: Spearman's non-parametric correlation test for IHC staining of 25 NSCLC patients.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Immunohistochemistry

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: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Phospho-proteomics, 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: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Stable Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Phospho-proteomics, 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) We used ProtoArray™ to identify EGFR as a novel interaction partner for AURKA. Briefly, expressed, recombinant his-tagged human AURKA was probed on ProtoArray™ human protein microarrays at seven concentrations (0, 0.5, 1.0, 5.0, 10, 50 and 100 ng/µl) in duplicate. It should be noted that EGFR-L861Q, but not EGFR-WT, was initially identified to interact with AURKA. (B) HEK293T were co-transfected with Myc-EGFR (EGFR-WT, EGFR-L858R and -L861Q mutants) and Flag-AURKA [AURKA-WT and AURKA-kinase dead (KD)]. After transfection for 48 hours, the cell extracts were subjected to co-immunoprecipitation with an anti-FLAG M2 affinity gel. Protein expression was determined by immunoblotting with anti-Myc and anti-FLAG antibodies. It showed that both EGFR-WT and mutated EGFR associates with AURKA-WT and AURKA-KD. Similar results were observed in at least three independent experiments. (C) The EGFR-AURKA interaction with or without EGF stimulation (10 ng/ml for 10 minutes) was determined via in situ PLA in A549 (EGFR-WT) and H1975 (EGFR-L858R-T790M mutations). Both EGFR-WT and -L858R mutant were associated with AURKA under EGF stimulation. The quantification of in situ PLA signals was shown on the right. (D) The phosphorylation sites of EGFR-Thr654 and -Ser1046 matched the substrate consensus sequences for AURKA. (E) H1975 cells showed more obvious phosphorylation at EGFR-Thr654, EGFR-Ser1046 and AURKA-Thr288 in nocodazole-arrested M-phase than in interphase.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Recombinant, Transfection, Immunoprecipitation, Expressing, Western Blot, In Situ, Mutagenesis, Phospho-proteomics

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: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Mutagenesis

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: Each column indicates a patient and each row indicates an antibody. The IHC intensity in the patients was defined as 0, 1 and 2 for AURKA, pEGFR-Thr654 and pEGFR-Ser1046 staining.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Staining

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: Wilcoxon two-sample t-test for IHC staining in 25 NSCLC patients.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Immunohistochemistry

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: Spearman's non-parametric correlation test for IHC staining of 25 NSCLC patients.

Article Snippet: Heat-induced epitope retrieval was performed in an antigen retrieval solution at 100°C for 10 minutes with a microwave and the sections were then incubated with a rabbit polyclonal anti-pEGFR-Thr654 antibody (1∶300 dilution, Abnova), rabbit polyclonal anti-pEGFR-Ser1046 antibody (1∶100 dilution, Abnova), and rabbit polyclonal anti-AURKA antibody (1∶400 dilution, Sigma-Aldrich).

Techniques: Immunohistochemistry