Journal: Molecular & Cellular Proteomics

Article Title: Bcl2-associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity

doi: 10.1074/mcp.m112.025882

Figure Lengend Snippet: FIG. 5. Validation of newly identified BAG3 interactors by co- immunoprecipitation. Immunoprecipitation assays of 14-3–3, ATG3, Hsp70, PSMB1, MAPK7, HSF1, and BAG3 proteins were carried out as described under “Experimental Procedures.” A, immu- noblotting demonstrated that BAG3 protein binds with 14-3–3, ATG3, PSMB1, MAPK7, HSF1, and Hsp70 proteins. No BAG3-inter- acting bands were observed in the negative control (IgG). “Input” stands for the total cell lysate extracted from HeLa cells. Similarly, reverse immunoprecipitation assays confirmed the binding of (B) 14-3–3, (C) ATG3, (D) PSMB1, (E) Hsp70, (F) MAPK7, and (G) HSF1 protein with BAG3.

Article Snippet: Purchased proteins were BAG3 (Abnova, Taipei, Taiwan), 14-3–3 , Hsp70, MAPK7, ATG3, PSMB1, HSF1, and ENO1 (OriGene Technologies, Rockville, MD).

Techniques: Biomarker Discovery, Immunoprecipitation, Negative Control, Binding Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via p90RSK-Mediated ERK5 S496 Phosphorylation

doi: 10.3389/fcvm.2018.00023

Figure Lengend Snippet: IR-induced p90RSK activation lead to ERK5 S496 phosphorylation, but not ERK5 TEY motif phosphorylation. ( A ) HUVECs were treated with IR by the indicated doses, and 30 min after IR, ECs were collected and p90RSK activity was detected by Western blotting with anti–p-p90RSK and anti-p90RSK antibodies. Representative images from three independent experiments are shown. ( B ) HAECs were pre-treated with FMK-MEA (5 µM) for 30 min and treated by IR (2 Gy) for the indicated times. ECs were collected, and total p90RSK, p90RSK phosphorylation, ERK5 S496 phosphorylation, ERK5 TEY motif phosphorylation, and total ERK5 were detected by Western blotting with anti-p90RSK, anti-phospho-p90RSK (S380), anti-phospho-ERK5 (S496), anti-phospho-ERK5 (TEY motif), and anti-ERK5. Representative images from three independent experiments are shown. ( C ) Quantification of IR-induced p90RSK activation (S380 phosphorylation; top), ERK5 S496 phosphorylation (middle), and ERK5 activation (TEY motif phosphorylation; bottom) is shown after normalization by total protein levels. Data represent mean ± SEM ( n = 3).

Article Snippet: Antibodies were purchased from the following vendors: anti-p90RSK1 (C-21, #SC-231) and anti-VCAM-1 (SC-8304) from Santa Cruz Biotechnology (Dallas, TX); anti-phospho-p90RSK (Ser380, #9341), anti-phospho-ERK5 (Thr218/Tyr220, #3371L), and anti-ERK5 (#3372) from Cell Signaling (Danvers, MA); anti-tubulin (T-5168) from Sigma (St. Louis, MO); anti-VE-cadherin (#555289) from BD Transduction Laboratory (San Diego, CA); and anti-phopho-specific ERK5 S496 antibody (#A02812) from Boster Bio (Pleasanton, CA).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via p90RSK-Mediated ERK5 S496 Phosphorylation

doi: 10.3389/fcvm.2018.00023

Figure Lengend Snippet: ERK5 S496 phosphorylation is critical for IR-induced EC inflammation. HUVECs were co-transfected with the NF-κB-Luc luciferase reporter vector and the pRL-CMV vector (renilla luciferase activity, internal control) together with the expression vector encoding the flag-tagged ERK5 wild type (WT) or the S496A mutant. Transfected cells were then exposed to gamma radiation (2 Gy) and harvested after 24 h. ( A ) NF-κB activation was quantified by measuring relative luciferase activity using a dual-luciferase reporter system. Shown are relative luciferase activity, presented as firefly luciferase/renilla luciferase activity ratio. ( n = 6) ( B ) VCAM1 expression was determined by measuring percentage of positive VCAM1-staining cells using the BD Accuri C6 Flow Cytometer system using the FL1 533/30 nm filter. ( n = 5)

Article Snippet: Antibodies were purchased from the following vendors: anti-p90RSK1 (C-21, #SC-231) and anti-VCAM-1 (SC-8304) from Santa Cruz Biotechnology (Dallas, TX); anti-phospho-p90RSK (Ser380, #9341), anti-phospho-ERK5 (Thr218/Tyr220, #3371L), and anti-ERK5 (#3372) from Cell Signaling (Danvers, MA); anti-tubulin (T-5168) from Sigma (St. Louis, MO); anti-VE-cadherin (#555289) from BD Transduction Laboratory (San Diego, CA); and anti-phopho-specific ERK5 S496 antibody (#A02812) from Boster Bio (Pleasanton, CA).

Techniques: Phospho-proteomics, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Control, Expressing, Mutagenesis, Activation Assay, Staining, Flow Cytometry

Journal: Frontiers in Cardiovascular Medicine

Article Title: Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via p90RSK-Mediated ERK5 S496 Phosphorylation

doi: 10.3389/fcvm.2018.00023

Figure Lengend Snippet: ERK5 S496 phosphorylation is critical for IR-induced apoptosis. HUVECs were transfected with the expression vector encoding the flag-tagged ERK5 wild type (WT) or the S496A mutant. Transfected cells were then exposed to gamma radiation (2 Gy), and harvested after 60 h. Cell apoptosis was determined by measuring percentage of Annexin V- positive cells (Abcam’s Annexin V-FITC Apoptosis Detection Kit) using the BD Accuri C6 Flow Cytometer system with the FL1 533/30 nm filter. Shown are mean ± SEM, n = 3–6.

Article Snippet: Antibodies were purchased from the following vendors: anti-p90RSK1 (C-21, #SC-231) and anti-VCAM-1 (SC-8304) from Santa Cruz Biotechnology (Dallas, TX); anti-phospho-p90RSK (Ser380, #9341), anti-phospho-ERK5 (Thr218/Tyr220, #3371L), and anti-ERK5 (#3372) from Cell Signaling (Danvers, MA); anti-tubulin (T-5168) from Sigma (St. Louis, MO); anti-VE-cadherin (#555289) from BD Transduction Laboratory (San Diego, CA); and anti-phopho-specific ERK5 S496 antibody (#A02812) from Boster Bio (Pleasanton, CA).

Techniques: Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Flow Cytometry

Journal: Türk Tarım ve Doğa Bilimleri Dergisi

Article Title: Ashwagandha root extract attenuates inflammation in Oleic acid induced-ALI/ARDS rat model via inhibition of ACE and MAPK signaling pathways

doi: 10.30910/turkjans.1209593

Figure Lengend Snippet: Figure 1. The effects of Ashwagandha on the MPO and MAPK levels on lung in ALI/ARDS. * denotes significant differences between other studied groups and control (*: p<0.05, ***: p<0.001), ε denotes significant differences between other studied groups and the OA group (ε: p<0.05, εε: p<0.01). Abbreviation used: OA: Oleic acid, Ash: Ashwagandha.

Article Snippet: The obtained supernatants were assayed for Myeloperoxidase (MPO), Glutathione (GSH), Superoxide Dismutase (SOD), Mitogen-activated protein kinases (MAPK), Angiotensin-converting enzyme (ACE) (BT LAB Company, China), and Total oxidant status (TOS) (Rel Assay Diagnostics, Gaziantep, Turkey) levels in rat lung tissue by enzyme-linked immunosorbent assay (ELISA) available kits in accordance with the manufacturer’s manuals.

Techniques: Control

Journal: Molecular cell

Article Title: Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m 6 A Methylation

doi: 10.1016/j.molcel.2020.10.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Active ERK5 , SignalChem , M32–10G.

Techniques: Control, Recombinant, Luciferase, Mutagenesis, Modification, Multiplex Assay, CRISPR, Generated, Ubiquitin Proteomics, Software