EMBO/sd-character-level-ner huggingface dataset:
Figure 6 (A) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E  (E) Proposed model of sequentially acting SOX proteins in neuronal and glial lineage specification. Silent neuronal, astrocyte and oligodendrocyte genes are all prebound by SOX3 in NPCs. In GPCs transactivating astrocytic enhancers, enriched for NFI motifs, (CRM A) become additionally targeted by SOX9 whereas non-activating astrocytic enhancers (CRMB) or CRMs around oligodendrocyte genes become pre-bound by SOX9 only. While SOX3 represses the activation of prebound genes, neuronal and glial gene expression become activated when SOX3 is downregulated and a cellular context of activating transcription factors has developed, which according to the model can consist of SOX11 in neurons, SOX10 in oligodendrocytes and NFIA and SOX9 in astrocytes. It should be noted that we do not provide any evidence that SOX3/SOX9 or NFIA/SOX9 physically interact or co-bind DNA
BICD1 expression in axons is upregulated by de-novo synthesis A) Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min.Figure 6Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min. Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells. (DMSO, grey: 100%, N=7 independent experiments; PPP, blue: 137.1 ± 7.3%, N=7 independent experiments; CHX, pink: 89.5 ± 12.7%, N=5 independent experiments; MG-132, teal: 124 ± 28.7, N=3 independent experiments; P=0.009, one-way ANOVA; Tukey's post-hoc test: DMSO-PPP * P=0.04, PPP-PPP+CHX * P=0.01; data shown are mean ± SEM). Schematic of puromycin-PLA assay. Cells were treated with DMSO, PPP or PPP + anisomycin for 30 min in the axonal compartment. After this puromycin was added to the medium for an additional 30 min. Cells were then fixed and the PLA protocol was performed (see Materials and Methods). Puromycin- Purple triangle, BICD1-Grey circles, Anti-puromycin antibody- green, Anti-BICD1 antibody- blue, Secondary antibodies- black. Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons. Quantification of puromycin-PLA signal. PPP causes an increase in PLA puncta in the axon of PMN, which is abolished by treatment with 40 µM anisomycin (DMSO, grey: 100%; PPP, blue: 139.3 ± 7.7%; PPP + anisomycin, purple: 86.3 ± 10.0; P=0.005, one-way ANOVA, Tukey's post-hoc test; DMSO vs PPP, * P=0.02; PPP vs PPP + anisomycin, ** 0.005, N=3 independent experiments, 27-57 images, data shown are mean ± SEM).
Decrease in the number of GFP‐Gal3 puncta is dependent on time and autophagy.Figure 2(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.
Figure 10Different forms coloured thus - cAMP-bound full-length (dark/light blue), cGMP-bound full-length (red), apo Bd1971∆EAL (yellow/orange) and half-occupied cAMP Bd1971∆EAL-mid (dark/light green). Residues from the opposing monomer labeled by a '. (A) Comparative conformational shifts when aligned on C-helix dimer. One monomer from the half-occupied form is empty (mid A) and adopts a similar pose to the apo form; the other monomer has bound cAMP (mid B) and adopts a pose halfway between the apo and bound forms. (B and C) The occupied cNMP monomer of Bd1971∆EAL-mid structure (green) possesses other features conformationally intermediate between the apo (yellow) and bound (blue) states, namely the position of M59 and the juxtaposition of the D63:R110 pair. (D) Comparison of nucleotide conformations between the three differing bound states of the Bd1971 cNMP sensor. The cAMP ligand in the Bd1971∆EAL-mid structure (green), is present in the syn form, with nucleotide base pointing toward the cyclic ribose phosphate. This conformation is like that observed for cGMP (red), but opposite to that of cAMP in the full-length (blue, occupied at both sites) complex. The interaction of R111' with the syn cAMP in Bd1971∆EAL-mid kinks R108 such that it cannot make a contact with the backbone carbonyl of I55, leaving M59 in upward conformation.
The effects of mutation of recognition motifs in wt Htt-552 on Htt-552 degradation.Figure 7(A) Mutation of Htt-552 of 18Q at 99KDRVN103 and 248NEIKV252. (B) Immunoprecipitation of exogenous expressed Hsc70 and Htt-552-18Q. Hella cells were transfected with wt and mutant Htt-18Q for 48 h. The lysates were subjected to immunoprecipitation with anti-Hsc70 antibody or no primary antibody. Mutation of Htt-552-18Q at 99-103 was labeled with ▴ and mutation of Htt-552-18Q at 248-252 was labeled with ▪. Ab: Antibody. (C) Inhibition of degradation of Htt-552-18Q with mutation of CMA recognition sites. The degradation of Htt-552-18Q in HeLa cells transiently transfected with human Hsc70 plasmid or vector control and Htt-552-18Q▴ was determined with Western blot analysis. Values are the mean±SE of three independent experiments. **P<0.01 (compared with the expression of Htt-552-18Q without treatment).
a) Wild-type yeast cultured in S(-NC) for 4 h.f4(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3) P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. ( (c) Labelling intensity of PtdIns(3) P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3) P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.
Figure 2FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA. C Removal of FBS from normal growth conditions (DMEM+10% FBS) results in MAGE-A3/6 protein degradation in HeLa cells. D, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA. F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL. Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. , H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.
a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.f4(a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.) 3′UTR of FoxO3 with scrambled (Scr) control (Ctrl), pre-miR-132 or pre-miR-212 (n=9). (b,c) Expression levels of FoxO3 on mRNA (b) and protein levels (c) in hearts of WT and α-MHC-miR-212/-132 transgenic (TG) mice. M: Size marker (n=9-13). (d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6-9; P-values against Scr+PE). (e-g) Expression levels of atrogin-1 (e) and Mcip1 (f) and calcineurin phosphatase activity levels (g) in hearts of WT and α-MHC-miR-212/132 TG mice (n=5-9). (h) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl, pre-miR-132 or pre-miR-212 (n=5). (i,j) Mcip1.4 (i) and atrogin-1 (j) mRNA levels in WT and miR-212/132 null (KO) mice 3 weeks after transaortic constriction (TAC) or Sham operation (n=5-7 per group). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. #P0.05 compared with WT TAC. FC, fold change.
Figure 4Ecto-5′-nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S::E5′NT (#303), Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) or Pro35S::mCherry (#305). E5′NT activity was measured after incubation with 100 µM of either ATP, ADP or AMP. In the membrane protein preparations from Pro35S::E5′NT (#303) lines phosphate release was specifically increased upon incubation with purines. Error bars represent the standard error of the mean from three technical repetitions. The coomassie stained SDS-PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines. Equal volumes were loaded. The experiment was repeated two times with similar results. Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry (#305) or Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) showing secretion of the E5′NT fusion protein. mCherry images show z-stacks of 14 image planes of 1 µm each. Scale bar=20 µm. The transgenic Arabidopsis line Pro35S::E5′NT (#303) expressing untagged full length SiE5′NT was better colonized by S. indica. Error bars of the qPCR data represent ±SE of the mean from three independent biological replicates. Asterisks indicate significance (Student's t-test, * p< 0.05). S. indica induced eATP release in different Arabidopsis transgenic lines. Culture medium was collected from mock treated or S. indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured. RLU: relative light units. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05). S. indica colonization of transgenic lines at 5 dpi. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05). Expression analysis of the eATP responsive gene At1g58420 measured by qRT-PCR. Error bars represent ±SE of the mean from three independent biological replicates (independent from those shown in Fig. 2C). Letters indicate significant groups (ANOVA, p<0.01, for the line 305 p<0.05).
Figure 4(A) Relative Quantitative Real-Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, student's t-test versus siNon-Target (siNT) treated cells, ***p < 0.001). (B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).
Immunohistochemical staining of liver sections for TGF-β and F4/80.Figure 6Liver sections from control and CCl4- and CCl4/R-III-treated mice were immunohistochemically stained for TGF-β and F4/80. Scale bar, 50 μm.
EPAC activation inhibits the Hla-dependent autophagy.Figure 3(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments. (B) CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (left panel) or myc-ΔEPAC (right panel). Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (C) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions (n = 20 cells/condition). These data are representative of two independent experiments
Figure 2A. Quantification of the mTFP/Venus fluorescence ratio of laconic (lactate sensor) showing concentration-dependent lactate uptake in HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM)) B. Representative images showing mTFP (blue) and Venus (yellow) fluorescence in control and lactate (20mM) treated HBVPs. C. Schematic illustration for the lactate uptake assay with 2 well chamber. Cells were seeded on a glass bottom dish. BMECs and adenoviral laconic-infected HBVPs were separated by 2 well chamber. After attachment, the 2 well chamber was removed for cell migration. D. Schematic illustration for the lactate uptake assay after chamber removal. At 24hr after chamber removal, the fluorescence of mTFP and Venus were observed in the middle of the wells (yellow area) E. Representative stitched image showing mTFP (green), Venus (yellow), DAPI (blue) and ERG1/2/3 (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. F. Representative images (high magnification; 60x) showing mTFP (cyan), Venus (yellow) and VECAD (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact. G. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs with endothelial contact (EC-PC contact) or HBVPs alone (PC only). (n=16 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) H. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or GLUT1 (lower panel) siRNA treated BMECs. VECAD immunostaining (purple) was used for endothelial staining. I. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or GLUT1 (siGLUT1) siRNA-treated BMECs. (n=12 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Identification of E4 as a modulator of retrograde axonal transport A) Small-molecule kinase inhibitor screen.Figure 1Small-molecule kinase inhibitor screen. Data are shown as an XY plot of normalized mean staining intensity of AlexaFluor555-HcT versus α-p75NTR. The α-p75NTR was detected using an AlexaFluor 647-conjugated donkey anti-rabbit secondary antibody [12]. Compounds that increased the accumulation of HcT or α-p75NTR by at least three standard deviations (yellow box) were classified as active compounds (A1-light blue, C3-purple, E4-dark blue). The negative control (EHNA) is shown in red (≥ 25 cell bodies were imaged per condition, N= 3 independent experiments). Speed distribution profile of PMN treated with 0.5 µM E4 or DMSO for 30 min. Graph shows an increase in the rate of axonal transport upon E4 treatment (DMSO, 153 endosomes, 13 axons; E4, 165 endosomes, 13 axons; N=4 independent experiments, data shown are mean ± SEM). PMN treated with 0.5 µM E4 for 30 min showed a significant decrease in phospho-IGF1R levels compared to controls. Protein levels were normalised to β-actin (*** P< 0.001, Students t-test, N=3 independent experiments, data shown are mean ± SEM).
Accumulation of α-Synuclein as a Consequence of Impaired Quality Control.Figure 2(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control. (B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.
Figure 7 Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology. (A) Evoked release upon action potential stimulation. Average EPSC amplitude (M18WT: 3.78 ± 0.58 nA, n=22; M18Y473F: 4.10 ± 0.61 nA, n=20, Mann-Whitney U test, p = 0.7915). Typical responses are depicted on the top. Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(B) Average mEPSC frequency (M18WT: 5.42 ± 1.86 Hz, n=17; M18Y473F: 4.42 ± 1.01 Hz, n=14, t-test with Welch correction, p = 0.6424), mEPSC amplitude (M18WT: 20.72 ± 0.7 pA, n=17; M18Y473F: 20,5 ± 0.9 pA, n=14, t-test, p = 0.8373) and mEPSC decay time (M18WT: 2.54 ± 0.20 ms, n=17; M18Y473F: 2.46 ± 0.18 ms, n=14, t-test, p = 0.7550). Example traces of spontaneous release are depicted on top. Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(C) EPSC depression during 10 Hz stimulation is normal. Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(D) Depression of EPSC charge during 40 Hz stimulation is normal. Inset shows the first 5 pulses. Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(E) EPSC amplitude recovery two seconds after an RRP depleting 40 Hz train. Average EPSC recovery (M18WT: 82.2 ± 6.7 %, n=17; M18Y473F: 65.5 ± 11.0 %, n=12, t-test, p = 0.1805). Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(F) The amount of DSE-triggered EPSC depression was quantified by the ratio of the average EPSC amplitude (taken over 4 pulses given at 0.2 Hz) before or immediately following 10 seconds depolarization to 0mV. Typical EPSCs before and following DSE induction are depicted on the left. Average DSE (M18WT: 0.57 ± 0.04 fold, n=20; M18Y473F: 0.53 ± 0.04 fold, n=16, t-test, p = 0.5227). Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(G) PDBu was bath applied during 0.05 Hz stimulation. Maximum potentiation of EPSC amplitude (M18WT: 2.56 ± 0.48 fold, n=7; M18Y473F: 2.82 ± 0.98 fold, n=8, t-test with Welch correction, p = 0.8168).
Figure 3A. Real-time PCR analysis of the genes coding for the pro-inflammatory cytokines IL-1β, IL-6, TNF-α in the hypothalamus of icv LPS injected mice at different times after injection (from 1h to 18h), normalized to values in icv saline injected mice (n=6 per group). B-E. Study of gene and protein hypothalamic expression of the chemokine CCL2 (B, C) in icv LPS injected mice. Real-time PCR analysis for CCL2 (B) at different times after injection (from 1h to 24h), normalized to values in control icv saline injected mice (n=6 per group). B-E. Study of gene and protein hypothalamic expression of the chemokine CCL2 (B, C) in icv LPS injected mice. at different times after injection (from 1h to 24h), normalized to values in control icv saline injected mice (n=6 per group). Measurement of CCL2 (C) ) concentration by EIA after icv LPS injection (black bars) or icv saline injection (grey bars) in mice at 1h, 6h and 18h after injection. B-E. Study of gene and protein hypothalamic expression of the MCH peptide (D, E) in icv LPS injected mice. Real-time PCR analysis for MCH (D) at different times after injection (from 1h to 24h), normalized to values in control icv saline injected mice (n=6 per group). B-E. Study of gene and protein hypothalamic expression of MCH peptide (D, E) in icv LPS injected mice. Measurement of MCH (E) concentration by EIA after icv LPS injection (black bars) or icv saline injection (grey bars) in mice at 1h, 6h and 18h after injection. Cerebellum was used as negative control (3 independent experiments, n=6 per group in each experiment). F. The decrease in MCH mRNA levels observed 18h after icv LPS injection is partly abolished by the CCR2 antagonist INCB3344 (10μM) and in CCR2 KO mice, as shown by real-time PCR analysis for MCH in the hypothalamus. Results were normalized to values in icv saline injected WT mice (n=6 per group). Data are expressed as means ± SEM. ** or $$ p<0.01. * saline vs icv LPS injection in WT mice. $ icv LPS injection in WT mice vs LPS+INCB3344 icv injection in WT mice or icv LPS injection in CCR2 KO mice.