Journal: Redox Biology

Article Title: An essential role for EROS in redox-dependent endothelial signal transduction

doi: 10.1016/j.redox.2024.103214

Figure Lengend Snippet: Effect of EROS knockdown on ERK1/2 phosphorylation and abundance of EROS, NOX2 and RAC1 A . Representative immunoblot shows ERK1/2 phosphorylation in response to histamine and VEGF stimulation in siControl transfected HUVEC, which is strongly attenuated in EROS knockdown cells. B . Statistical analysis of ERK1/2 phosphorylation (n = 3 for all conditions). C . Quantitative RT-PCR of HUVEC mRNA downregulating EROS reveals low EROS (yellow bar, n = 6), unchanged RAC1 (blue bar, n = 6) as well as NOX4 (gray bar, n = 6) and high NOX2 transcripts (red bar, n = 6). D . Western blots of protein lysates from HUVEC transfected with either siControl, siRAC1, siEROS or siNOX2 probed with antibodies for NOX2, RAC1, EROS and GAPDH. E-G . EROS, NOX2 and RAC1 are significantly downregulated within all single-guided siRNA samples, siEROS (yellow bars), siNOX2 (red bars) and siRAC1 (blue bars) compared to siControl (green bars). All values are presented as means ± SEM, **P < 0.01, ***P < 0.001 and ****P < 0.0001 compared to untreated siControl HUVEC, # P < 0.05 of EROS siRNA vs. NOX2 siRNA and ## P < 0.01 compared to same treatments, either histamine or VEGF, following siRNA knockdown of RAC1 vs. Control siRNA using 1-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Immunoblots for detection of p44/42 Mitogen-activated protein kinases specific phosphorylation sites (p-ERK1/2, Thr202/Tyr204) and phosphorylated endothelial nitric oxide synthase (p-eNOS, Ser1177) were incubated in 5 % BSA using respective phospho-specific primary antibodies (CST, Cell Signaling Technologies, Danvers, MA, USA) in a dilution of 1:1000.

Techniques: Western Blot, Transfection, Quantitative RT-PCR

Journal: Redox Biology

Article Title: An essential role for EROS in redox-dependent endothelial signal transduction

doi: 10.1016/j.redox.2024.103214

Figure Lengend Snippet: EROS knockdown induces reductive stress associated protein regulation resulting in diminished nitric oxide formation A . Heat map of critical siEROS and siRAC1 regulated shared proteins identified in functional analysis. B . Network analysis shows most significant protein interactions. The network has been refined with a highest confidence cut off of 0.900. Nodes are colored based on calculated fold change. C . Representative immunoblots show abundances of eNOS phosphorylation (p-eNOS, Ser1177), total eNOS (eNOS) and GAPDH following transfection with Control or EROS siRNA in HUVEC after stimulation with histamine or VEGF vs. untreated. D . Analysis of p-eNOS blots reveals p-eNOS Ser1177 phosphorylation in siControl HUVEC stimulated with histamine or VEGF for 10 min, which is blocked in EROS downregulated HUVEC. Total eNOS (gray open rhombic characters) is unchanged among samples. All values are presented as mean ± SEM, **P < 0.01 and ***P < 0.001 compared to untreated Control siRNA and # P < 0.05 or ### P < 0.001 compared to same treatments, either histamine or VEGF, of siEROS vs. siControl cells using 1way ANOVA. E . Average curves of NO ● imaging experiments in Control (black curve) and EROS (gray curve) siRNA-transfected cells stimulated with histamine and NOC-7 as indicated. F . Statistical analysis of Control (n = 27) and EROS siRNA-transfected (n = 27) cells indicates decreased NO ● levels following siRNA-mediated EROS knockdown, ****P < 0.0001 using unpaired t -test.

Article Snippet: Immunoblots for detection of p44/42 Mitogen-activated protein kinases specific phosphorylation sites (p-ERK1/2, Thr202/Tyr204) and phosphorylated endothelial nitric oxide synthase (p-eNOS, Ser1177) were incubated in 5 % BSA using respective phospho-specific primary antibodies (CST, Cell Signaling Technologies, Danvers, MA, USA) in a dilution of 1:1000.

Techniques: Functional Assay, Western Blot, Transfection, Imaging

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Postexercise muscle glycogen synthesis with glucose, galactose, and combined galactose-glucose ingestion

doi: 10.1152/ajpendo.00127.2022

Figure Lengend Snippet: Antibodies used in Western blot analysis

Article Snippet: Protein kinase B phosphorylation site Ser473 , Cell Signaling Technology , 4060 , 1:5,000.

Techniques: Western Blot, Concentration Assay, Phospho-proteomics

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Postexercise muscle glycogen synthesis with glucose, galactose, and combined galactose-glucose ingestion

doi: 10.1152/ajpendo.00127.2022

Figure Lengend Snippet: Antibodies used in Western blot analysis

Article Snippet: Protein kinase B phosphorylation site Thr308 , Cell Signaling Technology , 2965 , 1:5,000.

Techniques: Western Blot, Concentration Assay, Phospho-proteomics