Journal: Journal of proteome research

Article Title: A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T Cell Receptor and SLP-76 Complex

doi: 10.1021/acs.jproteome.6b01026

Figure Lengend Snippet: Quantitative phosphoproteomic analysis of receptor-proximal TCR signaling proteins. Heatmaps were calculated from five biological replicate experiments. Blue/yellow heatmaps represent temporal changes in phosphopeptide abundance in either the Jurkat PLC-γ1 null cell line (Jgamma1) or reconstituted cell line (Jgamma1.WT) following TCR stimulation. Black squares represent a phosphopeptide abundance equal to the geometric mean for that phosphopeptide across all time points for each cell line. Yellow and blue represent phosphopeptide abundances above and below the average, respectively. White dots in the yellow/blue heatmap squares indicate a statistically significant (q < 0.05) difference between the phosphopeptide abundance at the indicated timepoint and the lowest abundance of that peptide observed in the time course for that cell line. For each timepoint in which a sufficient number of replicate measurements were observed, the coefficient of variation (CV) was computed. The magnitude of the CV is indicated by the black/orange heatmap underneath each time point. Red/green heatmaps indicate the ratio between the peptide abundances measured in Jgamma1 and Jgamma1.WT cells for each phosphopeptide at each time point. Black signifies no difference in peptide abundance between the cell lines, while red and green represent reduced and elevated phosphopeptide levels in Jgamma1 relative to Jgamma1.WT, respectively. White dots on red/green ratio heatmap squares indicate a statistically significant difference (q < 0.05) in the comparison between Jgamma1 cells and Jgamma1.WT cells ratios for that time point.

Article Snippet: The PLC-γ1 deficient cell line Jgamma1 (ATCC#CRL-2678) and its reconstituted counterpart Jgamma1.WT (ATCC# CRL-2679) 14 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: Journal of proteome research

Article Title: A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T Cell Receptor and SLP-76 Complex

doi: 10.1021/acs.jproteome.6b01026

Figure Lengend Snippet: PLC-γ1 deficiency perturbs phosphorylation on canonical TCR signaling proteins. The canonical TCR signaling pathway is depicted, along with quantitative Jgamma1 to Jgamma1.WT ratio heatmaps depicting altered phosphorylation profiles of select sites. The interpretation of the heatmap color scheme as well as the white dots is the same as that described in Materials and Methods and Figure 1.

Article Snippet: The PLC-γ1 deficient cell line Jgamma1 (ATCC#CRL-2678) and its reconstituted counterpart Jgamma1.WT (ATCC# CRL-2679) 14 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: Journal of proteome research

Article Title: A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T Cell Receptor and SLP-76 Complex

doi: 10.1021/acs.jproteome.6b01026

Figure Lengend Snippet: Hierarchical clustering of known and predicted Lck substrates reveals PLC-γ1-dependent programs of phosphorylation. A) Clustered heatmap of phosphopeptide abundance ratios. The clustered data consists of 195 phosphopeptides in the data derived from proteins that are known or confidently predicted Lck substrates (see Materials and Methods for details). For each peptide selected, the ratios of Jgamma1.WT to Jgamma1 abundance at each timepoint were log2 transformed. Hierarchical clustering was performed using Pearson correlation coefficient as the distance metric and average linkage to calculate cluster distances. B) Time series ratios of phosphopeptides in the five largest clusters obtained. Each plot represents the time course of ratios for each peptide in the cluster (grey series) as well as the population average for the cluster (red series). The text to the right of each plot lists the KEGG-annotated T cell receptor signaling pathway components represented in the cluster, and the proportion of total phosphopeptides of each protein observed in the cluster.

Article Snippet: The PLC-γ1 deficient cell line Jgamma1 (ATCC#CRL-2678) and its reconstituted counterpart Jgamma1.WT (ATCC# CRL-2679) 14 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques: Derivative Assay, Transformation Assay

Journal: Journal of proteome research

Article Title: A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T Cell Receptor and SLP-76 Complex

doi: 10.1021/acs.jproteome.6b01026

Figure Lengend Snippet: Phosphorylation kinetics of tyrosine residues located on the T cell receptor ζ chain. Phosphorylation kinetics of TCRζ tyrosines A) Tyr83, B) Tyr111, C) Tyr123, D) Tyr142, and E) Tyr153 are depicted during early TCR signaling. Each data point represents the mean phosphopeptide abundance measured across five biological replicate experiments, and error bars indicate the standard error of the mean. Asterisks represent a statistically significant difference (q < 0.05) between Jgamma1 cells and Jgamma1.WT cells at the indicated time point.

Article Snippet: The PLC-γ1 deficient cell line Jgamma1 (ATCC#CRL-2678) and its reconstituted counterpart Jgamma1.WT (ATCC# CRL-2679) 14 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: Journal of proteome research

Article Title: A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T Cell Receptor and SLP-76 Complex

doi: 10.1021/acs.jproteome.6b01026

Figure Lengend Snippet: Phosphorylation kinetics of regulatory tyrosine residues on Lck, Zap-7, and Itk. Phosphorylation kinetics of Lck regulatory sites A) Tyr192, B) Tyr394, and C) Tyr505, Zap-7 D) Tyr319, E) Tyr492, F) Tyr493, and Itk G) Tyr512 are depicted during early TCR signaling. Each data point represents the mean phosphopeptide abundance measured across five biological replicate experiments, and error bars indicate the standard error of the mean. Asterisks represent a statistically significant difference (q < 0.05) between Jgamma1 cells and Jgamma1.WT cells at the indicated time point.

Article Snippet: The PLC-γ1 deficient cell line Jgamma1 (ATCC#CRL-2678) and its reconstituted counterpart Jgamma1.WT (ATCC# CRL-2679) 14 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: Journal of Nutritional Science

Article Title: Estimation of daily selenium intake by 3- to 5-year-old Japanese children based on selenium excretion in 24-h urine samples

doi: 10.1017/jns.2019.21

Figure Lengend Snippet: γ-Ray spectra of United States National Institute of Standards and Technology (NIST) Toxic Elements in Urine (SRM-2678) by pseudo-cyclic instrumental neutron activation analysis (PC-INAA).

Article Snippet: Fig. 1. γ-Ray spectra of United States National Institute of Standards and Technology (NIST) Toxic Elements in Urine (SRM-2678) by pseudo-cyclic instrumental neutron activation analysis (PC-INAA).

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Geranylgeraniol Suppresses the Expression of IRAK1 and TRAF6 to Inhibit NFκB Activation in Lipopolysaccharide-Induced Inflammatory Responses in Human Macrophage-Like Cells

doi: 10.3390/ijms20092320

Figure Lengend Snippet: GGOH modulates NFκB signaling molecules. Differentiated THP-1 cells were incubated with or without 10 µM GGOH for 24 h, then stimulated with LPS. The cells were harvested after the indicated time of LPS treatment, and protein levels were measured by western blot, as described in the experimental procedures ( A ). Relative protein levels of MyD88 ( B ), tumor necrosis factor receptor-associated factor 6 (TRAF6) ( C ), interleukin-1 receptor-associated kinase 1 (IRAK1) ( D ), TAK1 (E ), and IKKβ ( F ). All values represent the mean ± SEM; n = 3. * The values are significantly different compared with the corresponding control value at p < 0.05, as assessed by Student’s t-test.

Article Snippet: The membrane was blocked for 1 h with TBS-T (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween20) containing 5% bovine serum albumin (Sigma) and then incubated with antibodies against IκBα (#4814, Cell Signaling Technology), MyD88 (#3699, Cell Signaling Technology, Danvers, MA, USA), IRAK1 (#4359, Cell Signaling Technology), phosphorylated IRAK1 (Thr209; ab61799, Abcam, Tokyo, Japan), TAK1 (#4505, Cell Signaling Technology), phosphorylated TAK1 (Thr184/187; #4508, Cell Signaling Technology), IKKβ (#2678, Cell Signaling Technology), phosphorylated IKKα/β (Ser176/180; #2697, Cell Signaling Technology), TRAF6 (#4743, Cell Signaling Technology), NFκB p65 (#8242, Cell Signaling Technology), or phosphorylated NFκB p65 (Ser536; #3033, Cell Signaling Technology).

Techniques: Incubation, Western Blot, Control