Journal: Cell Reports

Article Title: Human Liver Memory CD8 + T Cells Use Autophagy for Tissue Residence

doi: 10.1016/j.celrep.2019.12.050

Figure Lengend Snippet:

Article Snippet: Percoll density gradient media , GE Healthcare , Cat# 17089101.

Techniques: Blocking Assay, Isolation, Recombinant, Saline, Staining, Luminex, Cell Isolation, Software, Imaging, Flow Cytometry

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 1. Western blot analysis of CFTR and STAT1 expression in un- treated (lane 1), PMA-treated (lane 2), TNF-treated (lane 3), and IFN- treated (lane 4) T84 (A) and RCMC (B). T84 cells were treated with PMA (10 ng/ml), human recombinant TNF (10 ng/ml), or IFN (10 ng/ml) for 24 h. RCMC were treated with PMA (10 ng/ml) or rat recombinant TNF (10 ng/ml) or IFN (10 ng/ml) for 24 h. Cell lysates were resolved on an 4 to 12% SDS-PAGE and blotted with anti-CFTR, anti-STAT1, or anti-actin antibody. Western blots shown represent three independent experiments. The effect of 24 h IFN treatment (80 ng/ml) on CFTR expression was characterized in T84 cells (C) and rat peritoneal mast cells (D), using confocal laser scanning microscopy (bar on the figure represents 10 m).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Western Blot, Expressing, Recombinant, SDS Page, Confocal Laser Scanning Microscopy

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 4. A, IFN-mediated up-regulation of mast cell CFTR protein requires activation of JAK2 and/or p38 and ERK. RCMC, LAD2, and T84 were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SB202190; 30 mg/ml), or ERK MAPK inhibitor (U0126; 30 mg/ml) for 24 h. Representative of three inde- pendent experiments. B, densitometry summary of data in Fig. 4. RCMC were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SP202190; 30 mg/ml), or ERK inhibitor (U0126; 30 mg/ml) for 24 h, and expression of CFTR (black columns) and STAT1 (gray columns) was analyzed by Western blot (n 3 independent experiments; error bars represent S.E.M.). As- terisks represent statistical significance as determined by Student’s t test (p 0.05).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Journal of neurochemistry

Article Title: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

doi: 10.1111/j.1471-4159.2007.04585.x

Figure Lengend Snippet: Fig. 2 Thr40 on EAAT4 is essential for transporter stimulation by S422DSGK1. Xenopus oocytes were injected with wild-type EAAT4, T40AEAAT4 or T504AEAAT4 alone or together with constitutively active S422DSGK1. Four days after cRNA injection, labeled L-glutamate uptake was studied (a) and western blotting of whole cell lysates performed (b). Arithmetic means ± SEM. * Indicates statistically sig- nificant difference to uptake in Xenopus oocytes expressing wild-type EAAT4 alone. + Indicates statistically significant difference to uptake in Xenopus oocytes expressing T504AEAAT4 alone. Uptake values were normalized to the mean value obtained in oocytes expressing wild-type EAAT4 alone.

Article Snippet: After blocking with 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.15% Tween 20 for 1 h at 19–21 C, blots were incubated overnight at 4 C with a rabbit Nedd4-2 antibody (diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-EAAT4 antibody (Alpha Diagnostic International, San Antonio, TX, USA, diluted 1 : 500 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit antiphospho(Ser/Thr) Akt/SGK substrate antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1 : 200 in TBS/0.15% Tween 20/5% bovine serum albumin), or a rabbit anti-GAPDH horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk).

Techniques: Injection, Labeling, Western Blot, Expressing

Journal: Journal of neurochemistry

Article Title: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

doi: 10.1111/j.1471-4159.2007.04585.x

Figure Lengend Snippet: Fig. 1 Disruption of putative SGK1 phosphorylation sites on EAAT4 abrogates transporter stimulation. Xenopus oocytes were injected with wild-type EAAT4 or SGK1-phosphorylation-site-deficient T40AT504AEAAT4 alone or together with constitutively active S422DSGK1. Four days after cRNA injection, labeled L-glutamate uptake was studied (a) and western blotting of whole cell lysates performed (b). Arithmetic means ± SEM. * Indicates statistically sig- nificant difference to uptake in Xenopus oocytes expressing wild-type EAAT4 alone. Uptake values were normalized to the mean value obtained in oocytes expressing wild-type EAAT4 alone.

Article Snippet: After blocking with 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.15% Tween 20 for 1 h at 19–21 C, blots were incubated overnight at 4 C with a rabbit Nedd4-2 antibody (diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-EAAT4 antibody (Alpha Diagnostic International, San Antonio, TX, USA, diluted 1 : 500 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit antiphospho(Ser/Thr) Akt/SGK substrate antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1 : 200 in TBS/0.15% Tween 20/5% bovine serum albumin), or a rabbit anti-GAPDH horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk).

Techniques: Disruption, Phospho-proteomics, Injection, Labeling, Western Blot, Expressing

Journal: Journal of neurochemistry

Article Title: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

doi: 10.1111/j.1471-4159.2007.04585.x

Figure Lengend Snippet: Fig. 3 Thr40 on EAAT4 is required for enhanced EAAT4 plasma membrane abundance by S422DSGK1. Surface EAAT4 expression was assessed by chemiluminescence in oocytes expressing EAAT4, T504AEAAT4 or T40AT504AEAAT4 alone or together with S422DSGK1. Arithmetic means ± SEM. *Indicates statistically significant difference to oocytes injected with EAAT4. + indicates statistically significant difference to surface abundance in Xenopus oocytes expressing T504AEAAT4 alone. Cell surface expression was normalized in each batch of oocytes to the mean relative light units value obtained in oocytes expressing EAAT4 alone.

Article Snippet: After blocking with 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.15% Tween 20 for 1 h at 19–21 C, blots were incubated overnight at 4 C with a rabbit Nedd4-2 antibody (diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-EAAT4 antibody (Alpha Diagnostic International, San Antonio, TX, USA, diluted 1 : 500 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit antiphospho(Ser/Thr) Akt/SGK substrate antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1 : 200 in TBS/0.15% Tween 20/5% bovine serum albumin), or a rabbit anti-GAPDH horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk).

Techniques: Clinical Proteomics, Membrane, Expressing, Injection

Journal: Journal of neurochemistry

Article Title: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

doi: 10.1111/j.1471-4159.2007.04585.x

Figure Lengend Snippet: Fig. 4 Disruption of putative SGK1 phosphorylation sites on EAAT4 abrogates transporter inhibition by Nedd4-2. Xenopus oocytes were injected with wild-type EAAT4, SGK1-phosphorylation-site-deficient T40AT504AEAAT4 (a) or phosphorylation-mimicking EAAT4 mutants (T40DT504DEAAT4, T40DEAAT4) (b) alone or together with the ubiquitin ligase Nedd4-2. Four days after cRNA injection, labeled L-glutamate uptake was studied. Arithmetic means ± SEM. * Indicates statistically significant difference to uptake in Xenopus oocytes expressing wild- type EAAT4 alone. Uptake values were normalized to the mean value obtained in oocytes expressing wild-type EAAT4 alone.

Article Snippet: After blocking with 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.15% Tween 20 for 1 h at 19–21 C, blots were incubated overnight at 4 C with a rabbit Nedd4-2 antibody (diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-EAAT4 antibody (Alpha Diagnostic International, San Antonio, TX, USA, diluted 1 : 500 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit antiphospho(Ser/Thr) Akt/SGK substrate antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1 : 200 in TBS/0.15% Tween 20/5% bovine serum albumin), or a rabbit anti-GAPDH horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk).

Techniques: Disruption, Phospho-proteomics, Inhibition, Injection, Ubiquitin Proteomics, Labeling, Expressing

Journal: Journal of neurochemistry

Article Title: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

doi: 10.1111/j.1471-4159.2007.04585.x

Figure Lengend Snippet: Fig. 5 Disruption of putative SGK1 phosphorylation sites on EAAT4 reduces transporter phosphorylation by S422DSGK1. Xenopus oocytes were injected with wild-type EAAT4, SGK1-phosphorylation-site-defi- cient mutants (T40AT504AEAAT4, T40AEAAT4) alone or together with S422DSGK1. Four days after cRNA injection, western blots were per- formed (a). Densitometric analysis (b). Phosphorylated EAAT4 / total EAAT4 band intensity values in oocytes co-expressing S422DSGK1 were normalized to the values obtained in oocytes expressing the transporter alone.

Article Snippet: After blocking with 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.15% Tween 20 for 1 h at 19–21 C, blots were incubated overnight at 4 C with a rabbit Nedd4-2 antibody (diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-EAAT4 antibody (Alpha Diagnostic International, San Antonio, TX, USA, diluted 1 : 500 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit antiphospho(Ser/Thr) Akt/SGK substrate antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1 : 200 in TBS/0.15% Tween 20/5% bovine serum albumin), or a rabbit anti-GAPDH horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk).

Techniques: Disruption, Phospho-proteomics, Injection, Western Blot, Expressing

Journal: Journal of neurochemistry

Article Title: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

doi: 10.1111/j.1471-4159.2007.04585.x

Figure Lengend Snippet: Fig. 6 Inhibition of intrinsic xNedd4-2 by RNAi up-regulates EAAT4 function. Xenopus oocytes were injected with EAAT4 alone or together with a double-stranded xNedd4-2 specific siRNA. 4 days after cRNA injection, xNedd4-2 silencing was assessed by western blotting (a) and labeled L-glutamate uptake determined (b). To demonstrate the specificity of the effect observed, the activity of the facilitative glucose transporter GLUT1 that is unaffected by Nedd4-2 was studied upon injection of xNedd4-2 siRNA oligo (c). Arithmetic means ± SEM.

Article Snippet: After blocking with 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.15% Tween 20 for 1 h at 19–21 C, blots were incubated overnight at 4 C with a rabbit Nedd4-2 antibody (diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit anti-EAAT4 antibody (Alpha Diagnostic International, San Antonio, TX, USA, diluted 1 : 500 in PBS/0.15% Tween 20/5% non-fat dry milk), a rabbit antiphospho(Ser/Thr) Akt/SGK substrate antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1 : 200 in TBS/0.15% Tween 20/5% bovine serum albumin), or a rabbit anti-GAPDH horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany, diluted 1 : 1000 in PBS/0.15% Tween 20/5% non-fat dry milk).

Techniques: Inhibition, Injection, Western Blot, Labeling, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Na V 1.7 α1 subunit expression in wild-type (WT) and R192Q KI TG cultures. (A) Representative immunofluorescent examples of Na V 1.7 α1 expression in WT and R192Q KI mouse trigeminal ganglia (TG) cultures. Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note extensive co-staining of Na V 1.7 α1 (red) and β-tubulin III (green) protein in both WT and KI samples. (B) Western blot example showing protein expression of Na V 1.7 α1 in TG from P12 mice. β-actin was used as a loading control. Histograms representing Na V 1.7 α1 relative optical density values for each group (* p = 0.46, Mann-Whitney test; n = 8 experiments). Note the significant difference between WT and KI groups. (C) The histograms quantify the cell diameter distribution of Na V 1.7 α1 immunofluorescence in different subgroups of WT and KI cultures ( n = 3 experiments). The percentages of positive neurons for Na V 1.7 were calculated by considering the total number of neurons labeled by β-Tubulin III as standard.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: Expressing, Staining, Western Blot, MANN-WHITNEY, Immunofluorescence, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Na V 1.7 α1 subunit expression in WT and R192Q KI TG cultures after pretreatment with ω-agatoxin IVA. (A) Representative immunofluorescent examples of Na V 1.7 α1 expression in WT and R192Q KI mouse TG cultures. ω-agatoxin IVA (200 nM, overnight: in this and subsequent Figures the toxin is abbreviated as ω-Agatoxin) was used to specifically block Ca V 2.1 channels (that are mutated in the KI model). Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note extensive co-staining of Na V 1.7 α1 (red) and β-tubulin III (green) protein in both WT and KI samples. (B) Histograms represent the percentage of Na V 1.7-positive cells in WT and KI cultures. Note significant difference between WT and KI control groups (* p = 0.015, Mann–Whitney test; n = 7 experiments), and the decrease in Na V 1.7 positive neurons in the KI after application of ω-agatoxin IVA ( p = 0.035; n = 3 experiments). (C) The histograms quantify the cell diameter distribution of Na V 1.7 α1 immunofluorescence in different subgroups in WT and KI ω-agatoxin IVA-treated cultures.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: Expressing, Blocking Assay, Staining, MANN-WHITNEY, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Co-expression of Na V 1.7 α1 subunit and P2X3R in WT and R192Q KI cultures. (A) Representative examples of Na V 1.7 α1–P2X3 immunostaining in WT and R192Q KI mouse TG cultures. Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note the extensive co-staining of Na V 1.7 α1 (red) and P2X3 (green) protein in both WT and KI samples. (B) Histograms represent percentage co-expression of Na V 1.7 α1–P2X3 proteins in WT and KI cultures ( p = 0.95, Mann–Whitney test; n = 3 experiments). (C) The histograms quantify the cell diameter distribution of Na V 1.7 α1–P2X3R immunofluorescence in different subgroups in WT and KI cultures.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: Expressing, Immunostaining, Staining, MANN-WHITNEY, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Na V 1.7 current in WT and R192Q KI TG neurons. (A) Mean amplitudes of the currents evoked by a square pulse 100 ms depolarizing step from –75 to –45 mV, in WT and R192Q KI TG neurons under control conditions and after application of 1 μM TTX. Note larger control current in KI (* p = 0.039, two-sample Student’s t -test). Number of cells in each group: n = 29 (WT, control), n = 20 (WT, TTX), n = 43 (KI, control), i = 24 (KI, TTX). (B) Representative traces of inward currents recorded from one WT neuron in response to the same stimulation in control and after 1 μM TTX; the trace of TTX-sensitive current was obtained by subtraction of the TTX-resistant current from the control. (C) Superimposed representative traces of WT and KI TTX-sensitive currents obtained by subtraction; note larger KI current. (D) Histograms represent the calculated mean amplitudes of the currents evoked by the same stimulus (100-ms step from –75 to –45 mV) in control and after Tp1a (7 nM, 30 min). *Indicates statistically significant change (two-sample Student’s t -test, p -values are given in the text body). Number of cells in each group: n = 29 (WT, control), n = 28 (WT, Tp1a), n = 43 (KI, control), n = 38 (KI, Tp1a). (E) Superimposed representative traces of WT and KI Tp1a-sensitive (Na V 1.7) currents obtained by subtraction of the TTX-resistant current from the control; note larger Na V 1.7 current in KI.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: