Journal: Molecular & Cellular Proteomics : MCP

Article Title: Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia *

doi: 10.1074/mcp.RA117.000419

Figure Lengend Snippet: Regulation of proteins after ischemia alters biological functions in neurons and BBB. A, Schema of the cell assembly, organization and interaction processes occurring in neurons of the infarct core (IC). B, Cell death in the cerebral cortex is activated in both neurons and BBB structures (in red, proteins found altered in both cell types). C, Upstream regulator predicted to be activated in neurons of the IC. Geometric red figures represent IC-up-regulated proteins taking part in these biological functions whereas green figures represent IC-downregulated proteins. Based on the state of the protein and their role in the process (arrow indicating activator, dash indicating inhibitor), the nature of several biological processes was predicted: orange indicates predicted activation andblue indicates predicted inhibition. Gray lines indicate direct relationship among proteins. ACTG1: actin, ADORA2A: adenosine receptor A2a, ALDOC: fructose-bisphosphate aldolase C, AP2A1: AP-2 complex subunit alpha-1, ATP1A3: sodium/potassium-transporting ATPase subunit alpha-3, ATP2B1: plasma membrane calcium-transporting ATPase 1, CAMKV: CaM kinase-like vesicle-associated protein, CFL1: cofilin-1, CRMP1: Dihydropyrimidinase-related protein 1, DPYSL2: Dihydropyrimidinase-related protein 2, EAA2: excitatory amino acid transporter 2, FBXO2: F-box only protein 2, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, GNAO1: guanine nucleotide-binding protein G(o) subunit alpha, GSN: gelsolin, HSPB1: heat shock protein beta-1, NAPB: beta-soluble NSF attachment protein, PACSIN1: syndapin-1, RAB3A: Ras-related protein Rab-3A, RPS3: 40S ribosomal protein S3, SEPT11: septin-11, SH3GL2: endophilin-A1, SYN1: synapsin-1.

Article Snippet: Slides were then incubated overnight at 4 °C with rabbit primary antibodies diluted in blocking buffer against adenosylhomocysteinase-2 (SAHH2 or AHCYL1, cat.#: ab178693, Abcam, Cambridge, UK), serine/arginine-rich splicing factor-1 (SRSF1 or SF2, cat.#: ab129108, Abcam), excitatory amino acid transporter-2 (EAA2 or GLT1, cat.#: NBP1–59632, Novus Biologicals, Littleton, CO), GTPase KRas (RASK or KRAS, cat.#: H00003845-M01, Novus; also cat.#: A1190, Neo Scientific, Woburn, MA), hepatocyte cell adhesion molecule (HECAM or HepaCAM, cat.#: sc-168078, Santa Cruz Biotechnology Inc., Dallas, TX), serrate RNA effector molecule homolog (SRRT or ARS2, cat.#: H00051593-B02P, Novus), protein-arginine deiminase type-2 (PADI2, cat.#: ab16478 and also ab50257, Abcam), cytochrome b-c1 complex subunit 7 (QCR7 or UQCRB, cat.#: ab190360, Abcam), NAD-dependent protein deacetylase sirtuin-1 (SIR2 or SIRT2, cat.#: H00022933-M01, Novus; also cat.#: A0273, Neo), neurotrimin (NTRI, cat.#: sc-390941, Santa Cruz), endophilin-A1 (SH3G2 or SH3GL2, cat.#: ab55702 and also ab169762, Abcam), and mitochondrial 4-aminobutyrate aminotransferase (GABT or ABAT, cat.#: ab81432, Abcam).

Techniques: Activation Assay, Inhibition, Binding Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia *

doi: 10.1074/mcp.RA117.000419

Figure Lengend Snippet: List of proteins altered in infarcted brain areas found by MS analysis exclusively in neurons, BBB structures or both. Bold proteins were selected as candidates for replication. FC: fold-change

Article Snippet: Slides were then incubated overnight at 4 °C with rabbit primary antibodies diluted in blocking buffer against adenosylhomocysteinase-2 (SAHH2 or AHCYL1, cat.#: ab178693, Abcam, Cambridge, UK), serine/arginine-rich splicing factor-1 (SRSF1 or SF2, cat.#: ab129108, Abcam), excitatory amino acid transporter-2 (EAA2 or GLT1, cat.#: NBP1–59632, Novus Biologicals, Littleton, CO), GTPase KRas (RASK or KRAS, cat.#: H00003845-M01, Novus; also cat.#: A1190, Neo Scientific, Woburn, MA), hepatocyte cell adhesion molecule (HECAM or HepaCAM, cat.#: sc-168078, Santa Cruz Biotechnology Inc., Dallas, TX), serrate RNA effector molecule homolog (SRRT or ARS2, cat.#: H00051593-B02P, Novus), protein-arginine deiminase type-2 (PADI2, cat.#: ab16478 and also ab50257, Abcam), cytochrome b-c1 complex subunit 7 (QCR7 or UQCRB, cat.#: ab190360, Abcam), NAD-dependent protein deacetylase sirtuin-1 (SIR2 or SIRT2, cat.#: H00022933-M01, Novus; also cat.#: A0273, Neo), neurotrimin (NTRI, cat.#: sc-390941, Santa Cruz), endophilin-A1 (SH3G2 or SH3GL2, cat.#: ab55702 and also ab169762, Abcam), and mitochondrial 4-aminobutyrate aminotransferase (GABT or ABAT, cat.#: ab81432, Abcam).

Techniques: Activity Assay, Protease Inhibitor, Binding Assay, Activation Assay

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: Primer sequences for quantitative polymerase chain reaction

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: Sequencing

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX restores the expression of EAAT2 after spinal cord injury. (A) Representative western blot of EAAT2 protein level in spinal cord tissues 28 days after injury. (B) Quantification of the protein expression of EAAT2. (C) EAAT2 mRNA expression in spinal cord tissues 28 days after injury. (D) Representative images of western blotting of EAAT2 in each group at 7 dpi. (E) Quantitative assessment of EAAT2 protein expression. (F) EAAT2 mRNA expression in each group at 7 dpi. (G, H) Representative figures and quantitative assessment of immunofluorescence staining for EAAT2 (red, Alexa Fluor 594), GFAP (green, Alexa Fluor 488), and DAPI (blue) in reactive astrocytes in each group at 7 dpi. In reactive astrocytes, the fluorescence intensity of EAAT2 in the SCI + Veh group was lower compared with the Sham group, whereas EAAT2 expression was increased in the SCI + RUX group compared with the SCI + Veh group. The arrow indicates the expression of EAAT2 in the processes of astrocytes. Scale bar: 10 µm. (I) Measurement of glutamate levels in injured tissues in each group at 7 dpi ( n = 3). (J) Representative western blot of p-JAK2, JAK2, p-STAT3, and STAT3 in each group at 7 dpi. (K, L) Quantification of p-JAK2/JAK2 and p-STAT3/STAT3 levels in each group. In B, C, E, F, H, I, K, and L, data were normalized by the sham group. Data are presented as the mean ± SD. The experiments were performed in triplicate. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). AS: Scar-forming reactive astrocytes; DAPI: 4′,6-diamidino-2-phenylindole; dpi: days post-injury; EAAT2: excitatory amino acid transporter 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; JAK2: Janus kinase 2; NA: naïve astrocytes; ns: not significant; p-JAK2: phosphorylated Janus kinase 2; p-STAT3: phosphorylated signal transducer and activator of transcription 3; RA: reactive astrocytes; RUX: ruxolitinib; SCI: spinal cord injury; STAT3: signal transducer and activator of transcription 3; Veh: vehicle.

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX rescues astrocyte EAAT2 loss and restores glutamate uptake in vitro. (A) The effect of RUX (0.5, 1, 1.5, 2 and 2.5 µM) on the viability of astrocytes was assessed via CCK-8 assay ( n = 3/ group). (B, C) Western blotting of EAAT2 expression in A1IM-treated astrocytes in each group pre-treated with RUX (0.2, 0.5 and 1 µM) as shown ( n = 3 per group). (D) Quantitative analysis of EAAT2 protein levels in each group ( n = 3/group). (E, F) Representative immunofluorescence staining images and quantification of EAAT2 (red, Alexa Fluor 594), GFAP (green, Alexa Fluor 488), and DAPI (blue) in primary mouse astrocytes for each group ( n = 3 per group). The A1IM group displayed lower fluorescence intensity of EAAT2 compared with the Control group, whereas the RUX group exhibited higher fluorescence intensity of EAAT2 compared with the A1IM group. Scale bar: 20 µm. (G) Relative glutamate uptake of astrocytes in each group ( n = 3). The experiments were conducted in triplicate. Data are normalized by the sham group. Data are presented as the mean ± SD. ## P < 0.01, vs . control group; ** P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). A1IM: A1-like astrocyte induction medium; C1q: complement component 1q; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; EAAT2: excitatory amino acid transporter 2; GFAP: glial fibrillary acidic protein; IL-1α: interleukin-1 alpha; ns: not significant; RUX: ruxolitinib; TNF-α: tumor necrosis factor-alpha.

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: In Vitro, CCK-8 Assay, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX restores EAAT2 loss in astrocytes by inhibiting the activation of STAT3 in vitro. (A) Representative western blotting of p-JAK2, JAK2, p-STAT3 and STAT3 expression of astrocytes in each group pre-treated with RUX (0.2, 0.5 and 1 µM) ( n = 3 per group). (B) Quantitative analysis of p-JAK2/JAK2 and p-STAT3/STAT3 level in each group. (C) Immunocytochemistry of GFAP (green, Alexa Fluor 488) and p-STAT3 (red, Alexa Fluor 594) in primary mouse astrocytes. DAPI (blue) was used to stain nuclei. The A1IM group exhibited higher nuclear p-STAT3 expression compared with the Control group, whereas the RUX group displayed decreased nuclear p-STAT3 expression compared with the A1IM group. Scale bar: 40 µm. (D) Quantitative results of relative p-STAT3 intensity. (E) Representative western blotting of STAT3 expression of astrocytes pre-treated with S3I-201 or vehicle. (F) Quantitative results of relative STAT3 expression level. (G) Primary mouse astrocytes were pretreated with S3I-201 (50 μM) or RUX for 1 hour and then exposed to LPS (1 µg/mL) or PBS for 5 hours. Western blotting was conducted to assess EAAT2 expression level, followed by quantitative analysis of EAAT2 protein expression (H). (I) Glutamate (100 nM) was introduced into each culture medium, and the relative glutamate uptake of astrocytes in each group was measured. Data are normalized to the control group. Data are presented as the mean ± SD ( n = 3 per group). ## P < 0.01, vs. control group; * P < 0.05, ** P < 0.01, vs. A1IM group (one-way analysis of variance followed by Tukey’s post hoc test). A1IM: A1-like astrocyte induction medium; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; EAAT2: excitatory amino acid transporter 2; GFAP: glial fibrillary acidic protein; JAK2: Janus kinase 2; ns: not significant; p-JAK2: phosphorylated JAK2; p-STAT3: phosphorylated STAT3; RUX: ruxolitinib; S3I-201: a STAT3 inhibitor; STAT3: signal transducer and activator of transcription 3.

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: Activation Assay, In Vitro, Western Blot, Expressing, Immunocytochemistry, Staining

Journal: Neuron

Article Title: Astrocyte-secreted chordin like 1 drives synapse maturation and limits plasticity by increasing synaptic GluA2 AMPA receptors

doi: 10.1016/j.neuron.2018.09.043

Figure Lengend Snippet: (A-G) Chrdl1 KO mice display altered mEPSC kinetics in the developing visual cortex at P14. (A) Example mEPSC recordings from layer 2/3 pyramidal neurons in acute visual cortex slices. (B) Average mEPSC from all recordings aligned to rise time (left) and expanded timescale (right). (C-G) Quantification of mEPSCs: Chrdl1 KO mEPSC kinetics are altered, with increased decay time (C), rise time (D) and area (E); mEPSC frequency (F) and amplitude (G) are not altered. N=10 cells WT, 11 cells KO. (H-K) Astrocyte glutamate transporters are not significantly altered at synapses in Chrdl1 KO mice. (H) Example images of WT (top) and Chrdl1 KO (bottom) P14 visual cortex sections immunostained for GLT1 and VGlut2 along with Aldh1l1-GFP to mark astrocytes. (I-K) Quantification of immunostaining: Chrdl1 KO shows significant decrease in presynaptic VGlut2 puncta (K), no change in GLT1 on astrocytes (J) and a significant decrease in GLT1 colocalized with VGlut2 (I). N=3 WT, 3 KO mice. (L-N) No alteration in spine density in layer 2/3 neurons at P28 in Chrdl1 KO. (L) Example images of dendrites and spines visualized using Golgi staining. (M) Quantification of all dendritic protrusions, normalized per μm dendrite. (N) Quantification of spine types, normalized per μm dendrite. N=5 WT, 5 KO mice. Bar graphs mean±s.e.m., with individual data points representing cells (C-G) and mice (I-K, M-N). Statistics by T-test, significance as stated on graph. See also Figure S4,S5,S6; Tables S2,S3,S4.

Article Snippet: Primary antibodies used were guinea pig anti-VGlut2 (Millipore AB2251) at 1:1000 dilution, and rabbit anti-EAAT2/GLT1 (Novus Biologicals NBP1–20136SS) at 1:300 dilution.

Techniques: Immunostaining, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

doi: 10.3892/etm.2014.2024

Figure Lengend Snippet: TNF-α treatment elevates the expression of EAAT2 in the brain. (A) Expression levels of the EAATs following TNF-α treatment. (B) Quantification of the expression levels of the EAATs. Data are presented as the mean ± standard deviation and were analyzed by one-way analysis of variance (n=3). ** P<0.01 compared with saline controls. TNF-α, tumor necrosis factor-α; EAAT, excitatory amino-acid transporter.

Article Snippet: The membranes were incubated at 4°C overnight with antibodies against EAAT1 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China), EAAT2 (1:500; Wuhan Boster Biological Technology, Ltd.), GFAP (1:2,000) or β-actin (1:1,000; Cell Signaling Technology, Inc.) diluted in blocking solution.

Techniques: Expressing, Standard Deviation, Saline

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

doi: 10.3892/etm.2014.2024

Figure Lengend Snippet: Expression of EAATs in astrocytes treated with different doses of TNF-α. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression of EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=5). * P<0.05 and ** P<0.01 compared with the control group (0 ng/ml TNF-α). TNF-α, tumor necrosis factor-α; EAAT, excitatory amino-acid transporter; GFAP, glial fibrillary acidic protein.

Article Snippet: The membranes were incubated at 4°C overnight with antibodies against EAAT1 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China), EAAT2 (1:500; Wuhan Boster Biological Technology, Ltd.), GFAP (1:2,000) or β-actin (1:1,000; Cell Signaling Technology, Inc.) diluted in blocking solution.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

doi: 10.3892/etm.2014.2024

Figure Lengend Snippet: Expression levels of EAATs in astrocytes treated with TNF-α for different time-periods. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression levels of the EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=6). ** P<0.01 compared with the control group (prior to TNF-α treatment, 0 h). TNF-α, tumor necrosis factor-α; EAAT, excitatory amino-acid transporter; GFAP, glial fibrillary acidic protein.

Article Snippet: The membranes were incubated at 4°C overnight with antibodies against EAAT1 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China), EAAT2 (1:500; Wuhan Boster Biological Technology, Ltd.), GFAP (1:2,000) or β-actin (1:1,000; Cell Signaling Technology, Inc.) diluted in blocking solution.

Techniques: Expressing, Western Blot, Standard Deviation, Control