Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 1. VEGF and EGF stimulate differential ERK5 phosphorylation. HDMECs and HeLa cells were serum-starved overnight prior to stimulation for 10 min with either VEGF (50 ng/mL) or EGF (50 ng/mL), respectively. Cells were lysed in RIPA buffer, and lysates were resolved by 7.5–10% SDS-PAGE or Phos-tag SDS-PAGE and analysed by Western blotting. Results are from one experiment representative of three independent experiments.

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, SDS Page, Western Blot

Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 2. VEGF does not stimulate C-terminal phosphorylation of ERK5 in HDMECs. (A) HDMECs and HeLa cells were serum-starved overnight prior to stimulation for various time periods with either VEGF (50 ng/mL) or EGF (50 ng/mL), respectively. Cells were lysed in RIPA buffer and lysates were resolved by Phos-tag SDS-PAGE and analysed for ERK5 activation using site-specific phospho-antibodies. Protein loading was confirmed by blotting for Actin. (B) Quantification of ERK5 level by densitometric analysis. Data are shown as mean normalised to Actin level (n = 2). (C) Quantification of phospho-ERK5 (T218/Tyr220) level by densitometric analysis. Data are shown as mean normalised to Actin level (n = 2). (D) Quantification of phospho-ERK5 (Thr732) level by densitometric analysis. Data are shown as mean normalised to Actin level (n = 2).

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, SDS Page, Activation Assay

Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 3. Analysis of the canonical ERK5 signalling axis in HDMECs and HeLa cells. (A) HD- MECs or (B) HeLa cells were transfected with siRNA duplexes (10 nM) targeting MEKK2, MEKK3, MEK5, and ERK5 or non-silencing (N.S.) siRNA. Then, 24 h after transfection, cells were serum- starved overnight and then stimulated with VEGF (50 ng/mL) or EGF (50 ng/mL) for 10 min. Cells were lysed in RIPA buffer and lysates were resolved by SDS-PAGE and analysed by Western blotting. (C,D) Quantification of phospho-ERK5 (Thr218/Tyr220) level by densitometric analysis. Data are shown as mean fold change in relation to basal (untransfected cells) normalised to Actin level ± SEM (n = 3). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test, where * p < 0.05 and ** p < 0.01, ns = not significant.

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Transfection, SDS Page, Western Blot

Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 5. VEGF-mediated ERK5 activation regulates AKT phosphorylation in endothelial cells. (A) HDMECs or (B) HeLa cells were serum-starved overnight and then treated with either vehicle control (V.C.; 0.1% DMSO), or the MEK5 inhibitor BIX02189 (1 µM), or the ERK5 inhibitors AX15836 (1 µM) or JWG071 (1 µM) for 1 h prior to stimulation with either VEGF (50 ng/mL) or EGF (50 ng/mL) for 10 min. Cells were lysed in RIPA buffer and lysates were resolved by 10% SDS-PAGE or Phos- tag SDS-PAGE and analysed by Western blotting. (C) Quantification of HDMECs Phos-tag ERK5 level by densitometric analysis. Data are shown as mean fold change relative to basal (vehicle control) normalised to Actin level ± SEM (n = 3). (D) Quantification of HeLa Phos-tag ERK5 level by densitometric analysis. Data are shown as mean fold change relative to basal (vehicle control) normalised to Actin level ± SEM (n = 3). (E) Quantification of HDMECs Phos-AKT level by densitometric analysis. Data are shown as mean fold change relative to basal (vehicle control) normalised to Actin level ± SEM (n = 3). (F) Quantification of HeLa Phos-AKT level by densitometric analysis. Data are shown as mean fold change relative to basal (vehicle control) normalised to Actin level ± SEM (n = 3). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test, where ** p < 0.01, ns = not significant.

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Phospho-proteomics, Control, SDS Page, Western Blot

Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 6. VEGF stimulation results in cytosolic and plasma membrane localised ERK5, which co- localises with phosphorylated AKT in endothelial cells. (A) HDMECs or (B) HeLa cells were serum-starved overnight and then treated with media (Basal) or stimulated with either VEGF (50 ng/mL) or EGF (50 ng/mL) for 10 min or 30 min. Cells were then fixed in paraformalde- hyde, permeabilised, and stained with an antibody to ERK5 (green), phospho-AKT Ser373 (magenta) or Hoechst (blue) and analysed by immunofluorescence. Areas of pixel intensity correlation are shown as white/grey in the merged images. Scale bar = 10 µm. Results are from one experiment representative of three independent experiments.

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 7. VEGF-mediated ERK5 activity is not required for MEF2 transcriptional activity in en- dothelial cells. (A) HDMECs and HeLa cells were serum-starved overnight and then treated with either vehicle control (0.1% DMSO) or the MEK5 inhibitor BIX02189 (BIX; 1 µM) or the p38 MAPK inhibitor BIRB796 (BIRB; 1 µM), for 1 h prior to stimulation with VEGF (50 ng/mL) or EGF (50 ng/mL) for 10 min. Cells were lysed in RIPA buffer, and lysates were resolved by 10% SDS-PAGE or Phos-tag

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Control, SDS Page

Journal: Cells

Article Title: VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

doi: 10.3390/cells12060967

Figure Lengend Snippet: Figure 8. Schematic diagram of differential ERK5 activation by VEGF in endothelial cells and EGF in HeLa cells. Ligand-mediated activation of EGFR-1 in HeLa cells activates MEK5 predomi- nantly via MEKK2, resulting in the phosphorylation of ERK5 on the activation-loop T-E-Y motif in the ERK5 kinase domain, resulting in a conformational change in ERK5 and facilitating phosphorylation of ERK5 on multiple residues in the C-terminal transcriptional transactivation domain (TAD). The nuclear localisation sequence (NLS) within the C-terminal domain is now able to promote a nuclear localisation of ERK5. ERK5 is thought to drive MEF2-dependent gene expression by both directly phosphorylating MEF2 transcription factors and by the involvement of the ERK5 TAD. In contrast, ligand-mediated activation of VEGFR-2 in endothelial cells activates MEK5 via MEKK3, resulting in the phosphorylation of ERK5 on the activation-loop T-E-Y motif in the ERK5 kinase domain. However, ERK5 does not undergo phosphorylation in the C-terminus and is unable to translocate to the nucleus, and is instead localised to the cytoplasmic and plasma membrane area ultimately allowing phosphorylation of AKT and suppression of apoptosis in endothelial cells. In contrast to EGFR-1 signalling, MEF2 transcriptional activity in response to VEGFR-2 activation proceeds via p38 MAPK.

Article Snippet: Human epithelial cervical cancer cell line (HeLa) were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Phospho-proteomics, Sequencing, Gene Expression, Clinical Proteomics, Membrane, Activity Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Phosphopeptide Enrichment Using Offline Titanium Dioxide Columns for Phosphoproteomics

doi: 10.1007/978-1-62703-360-2_8

Figure Lengend Snippet: Nanoflow reversed-phase liquid chromatography coupled online with a linear ion trap mass spectrometer for the analysis of TiO2-enriched phosphopeptides from a HeLa cell lysate using collision-induced dissociation (CID) for tandem MS and phosphate neutral loss-dependent MS/MS/MS (RPLC-MS2-MS3). The analysis is for 5 μL injection of phosphopeptides onto a 75 μm ID capillary column. The lower panel is the base peak chromatogram of the analysis. The upper panel shows the MS3 or MS2 spectra of the phosphopeptides ALVAT*PGKK, SGAQASSTPLS*PTR, and LLT*PTHSFLAR identified, respectively, from nucleolin, lamin-A/C, and microtubule-associated protein 7. The relatively high abundance levels of the LC chromatographic peaks, from which these phosphopeptides were eluted, demonstrate that phosphopeptides were effectively enriched from the HeLa cell lysate. The asterisk marks the phosphorylated Ser or Thr residues.

Article Snippet: Cell Culture of HeLa Cells Human cervical epithelial cancer cell line HeLa (ATCC).

Techniques: Reversed-phase Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Injection

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Phosphopeptide Enrichment Using Offline Titanium Dioxide Columns for Phosphoproteomics

doi: 10.1007/978-1-62703-360-2_8

Figure Lengend Snippet: Nanoflow reversed-phase liquid chromatography coupled online with a linear ion trap mass spectrometer for the analysis of TiO2-enriched phosphopeptides from a HeLa cell lysate using collision-induced dissociation (CID) for tandem MS and phosphate neutral loss-dependent MS/MS/MS (RPLC-MS2-MS3). The analysis is for 5 μL injection of phosphopeptides onto a 75 μm ID capillary column. The lower panel is the base peak chromatogram of the analysis. The upper panel shows the MS3 or MS2 spectra of the phosphopeptides ALVAT*PGKK, SGAQASSTPLS*PTR, and LLT*PTHSFLAR identified, respectively, from nucleolin, lamin-A/C, and microtubule-associated protein 7. The relatively high abundance levels of the LC chromatographic peaks, from which these phosphopeptides were eluted, demonstrate that phosphopeptides were effectively enriched from the HeLa cell lysate. The asterisk marks the phosphorylated Ser or Thr residues.

Article Snippet: Human cervical epithelial cancer cell line HeLa (ATCC).

Techniques: Reversed-phase Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Injection