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

Journal: FEBS letters

Article Title: Neuronal glutathione depletion elevates the Aβ42/Aβ40 ratio and tau aggregation in Alzheimer's disease mice.

doi: 10.1002/1873-3468.14895

Figure Lengend Snippet: Fig. 3. Protein levels of presenilin1 and APP-related fragments. (A, B) The protein levels of presenilin1 and APP-related fragments were determined in the cortex of 4- and 8-month-old WT/GCLC-KO (A) and 4- and 12-month-old AppNL-G-F/AppNL-G-F X GCLC-KO (B) mice via western blotting using antibodies against APP, presenilin1 (PS1)-NTF, and PS1-CTF. The fraction utilized for Western blotting is described in the Materials and Methods section. The values shown in the graph represent the band intensity of each protein divided by the intensity of GAPDH or b-actin, and expressed as the mean level SEM (n = 3 or 4, *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). Groups were compared using Student’s t-test for significance. In 12-month-old mice, the band intensity was normalized to the intensity of the b- actin band, because the protein levels of GAPDH were significantly altered in GCLC-KO mice. Figures 3A,B and 4A depict Western blots conducted under identical conditions, and hence the GAPDH and b-actin loading controls are the same between these figures.

Article Snippet: Product # Dilution IHC WB Ab (N1D) Saido et al., J Biol Chem, 1994 [48] 1 : 500 AT8 (pS202/pT205-Tau) Innogenetics #90206 (anti PHF-TAU) 1 : 200 Neprilysin Leuca #NCL-L-CD10-270 1 : 200 Neprilysin R&D Systems #AF1126 1 : 500 APP Merck Millipore #MAB348 (clone 22c11) 1 : 2000 Presenilin-1 NTF (G1Nr5) Tomita et al., J Neurosci, 1999 [49] 1 : 2000 Presenilin-1 CTF (G1L3) Tomita et al., J Neurosci, 1999 [49] 1 : 2000 Tau5 (total Tau) Thermo #AHB0042 (Tau5) 1 : 1000 GAPDH Abcam #ab8245 1 : 10 000 b-Actin Sigma #A5441 1 : 5000 4 FEBS Letters (2024) a 2024 The Author(s).

Techniques: Western Blot