Journal: Molecular medicine reports

Article Title: Saururus chinensis (Lour.) Baill. extract promotes skeletal muscle cell differentiation by positively regulating mitochondrial biogenesis and AKT/mTOR signaling in vitro .

doi: 10.3892/mmr.2024.13250

Figure Lengend Snippet: Figure 6. SCE regulates AKT/mTOR and its downstream effectors in myotubes. (A) C2C12 myoblasts were induced to differentiate in a DMEM containing 2% horse serum, treated for 5 days with different concentration of SCE (1, 5 and 10 ng/ml) and analyzed using western blotting (left). Quantification of protein expression levels (n=3 per group) (right). **P<0.01 and ***P<0.001 vs. the control. (B) Cells were treated with 10 ng/ml SCE in DM for 1, 3 and 5 days and western blotting was performed using the indicated antibodies (left). Quantification of protein expression levels (n=3 per group) (right). *P<0.05, **P<0.01 and ***P<0.001 vs. the DMSO; #P<0.05, ##P<0.01 and ###P<0.001 vs. the DMSO at the indicated time points. SCE, Saururus chinensis (Lour.) Baill. extract; p‑, phosphorylated; p70S6K1, ribosomal protein S6 kinase B1.

Article Snippet: Specific antibodies against myosin heavy chain (MyHC; 1:500; cat. no. sc‐376157), myogenic differen‐ tiation 1 (MyoD; 1:1,000; cat. no. sc‐377460), myogenin and peroxisome proliferator‐activated receptor‐gamma coactivator‐1 α (Pgc‐1α; 1:1,000; cat. no. sc‐518038) were obtained from Santa cruz Biotechnology, inc. antibodies against non‐phospho (active) β‐catenin (1:1,000; cat. no. 8814), β‐catenin (1:1,000; cat. no. 9582), phospho‐aMPK (1:1,000; cat. no. 2535), aMPK (1:1,000; cat. no. 2532), phospho‐AKT (1:1,000; cat. no. 9271), AKT (1:1,000; cat. no. 9272), phospho‐mTOR (1:1,000; cat. no. 2971), mTOR (1:1,000; cat. no. 2983), phospho‐ribosomal protein S6 kinase B1(p70S6K1) (1:1,000; cat. no. 9234), p70S6K1 (1:1,000; cat. no. 2708) and glyceraldehyde 3‐phos‐ phate dehydrogenase (gaPdH; 1:1,000; cat. no. 2118) were purchased from Cell Signaling Technology, Inc.

Techniques: Concentration Assay, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: (A) Cells expressing either ERBB4 (upper panel), ERBB4 and ERBB3 (middle panel) or ERBB3 receptors (lower panel) were cultured in medium without ligand, in the presence of 10 ng/ml IL3, or in the presence of 10 or 100 ng/ml NRG1. Cell numbers were counted at the indicated time points. (B) Parental Ba/F3 or Ba/F3 cells expressing ERBB3, ERBB4 or both receptors were incubated in the absence of IL3 and treated with 100 ng/mL NRG1 where indicated. Total cell lysate were then prepared and immunoblotted with phosphoepitope- and protein-specific antibodies for ERBB3, ERBB4, Akt and MEK.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Expressing, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Ba/F3 ERBB3/ERBB4 or ERBB4 cells were treated in the three replicate experiments as indicated. Upon lysis and proteolytic digestion, peptides were differentially labeled with the three isotopic variant of mTRAQ and then pooled prior to peptide separation by high pH reversed phase chromatography and IMAC phosphopeptide enrichment. Phosphopeptide fractions were then analyzed by quantitative LC-MS on a LTQ Orbitrap Velos instrument. Lower panel: Characteristic mTRAQ patterns shown for a peptide harboring a NRG1-induced phosphosite in ERBB3/ERBB4 cells, which less strongly up-regulated in the absence of ERBB3 in ERBB4-expressing Ba/F3 cells.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Lysis, Labeling, Variant Assay, Reversed-phase Chromatography, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: (A) Volcano plot of NRG1-regulated phosphorylation in Ba/F3 cells expressing ERBB3 and ERBB4. (B) Volcano plot comparison of phosphorylation sites in NRG1-treated ERBB3/ERBB4 versus NRG1-treated ERBB4 expressing Ba/F3 cells. In both comparisons, log 10 -transformed, average phosphosite ratios are plotted against their standard deviations determined from mTRAQ replicate quantifications. Significantly regulated class I sites according to the Global Mean Rank test are depicted in red, all other sites in blue. The dashed grey lines indicate two-fold regulation.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Phospho-proteomics, Expressing, Comparison, Transformation Assay

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Scatter plot of the mean ERBB3/ERBB4 ± NRG1 ratios with mean ERBB3/ERBB4 versus ERBB4 ratios from NRG1-treated cells. Reproducibly quantified ERBB3 phosphosites are encircled.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques:

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Selected phosphorylation sites induced by NRG1 treatment.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Phospho-proteomics, Sequencing