Journal: Brain research bulletin

Article Title: Hindbrain Dorsal Vagal Complex AMPK Controls Hypothalamic Gluco-regulatory Transmitter and Counter-Regulatory Hormone Responses to Hypoglycemia

doi: 10.1016/j.brainresbull.2018.11.016

Figure Lengend Snippet: Impact of Cc Pretreatment on Ventromedial Hypothalamus Nucleus (VMN) AMPK and, Glutamate Decarboxylate65/67 (GAD65/67), and Neuronal Nitric Oxide Synthase (nNOS) Protein Expression during Insulin-Induced Hypoglycemia (IIH). Results show mean normalized VMN AMPK (Panel A), pAMPK (Panel B), GAD65/67 (Panel C), and nNOS (Panel D) O.D. values + S.E.M. for V/V, V/INS, and Cc/INS treatment groups (n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Proteins of interest were probed with primary polyclonal antisera raised in rabbit against AMPK α1/2 (1:2,000; 2532s; Cell Signaling Technology, Danvers, MA), pAMPK α1/2 (Thr 172; 1:2,000; 2535; Cell Signaling Technol.), RFamide related peptide-1 (RFRP-1; 1:1,000, sc-67010; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or neuronal nitric oxide synthase (nNOS; 1:2,000, NBP1–39681; Novus Biologicals, LLC, Littleton, CA), or raised in goat against NPY (1:2,500, NBP1–46535; Novus Biol.), POMC (1:2,000, NB100–1533; Novus Biol.), GAD 65/67 (1:2,000, AB1511; EMD Millipore, Burlington, MA), RFamide related peptide-3 (RFRP-3; 1:1,000, sc-32380; Santa Cruz Biotechnol.), or ORX-A (1:1,000, sc-8070; Santa Cruz Biotechnol.).

Techniques: Expressing

Journal: Cancers

Article Title: Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression.

doi: 10.3390/cancers17030477

Figure Lengend Snippet: Figure 3. Effects of PGE2 and COX-2 inhibition on nNOS expression and NO levels in hu- man melanoma cells. (a) A375 cells were treated with PGE2 (25 µM) with and without IFN-γ (250 units/mL) for 24 h, followed by immunoblot analysis of nNOS expression. β-actin was used to normalize loaded protein. Full-length blots of manuscript is shown in Figure S5. (b) Representative flow cytometry images of intracellular NO levels increased by PGE2 in the presence and absence of IFN-γ as detected using a DAF fluorescence probe. (c) Immunoblot analysis of nNOS expression levels after celecoxib treatment (50 µM) for 48 h. Full-length blots of manuscript is shown in Figure S6. (d) Celecoxib cotreatment reduced intracellular NO levels in the presence of IFN-γ in A375 cells. The impact of different treatments on NO levels in human melanoma SK-MEL-28 cells is shown in Supplementary Figure S10. * p < 0.05, ** p < 0.01, ns—not significant.

Article Snippet: Mouse monoclonal anti-human nNOS (#MAB2416; Novus Biologicals, Centennial, CO, USA), mouse monoclonal anti-human β-Actin (#8H10D10; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-human PD-L1 (#E1L3N; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-human COX-2 (#D5H5; Cell Signaling Technology, Danvers, MA, USA) antibodies were used as primary antibodies, and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit (1:5000; 1:2000; Cell Signaling Technology, Danvers, MA, USA) were utilized as secondary antibodies for western blotting analysis.

Techniques: Inhibition, Expressing, Western Blot, Flow Cytometry, Fluorescence

Journal: Cancers

Article Title: Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression.

doi: 10.3390/cancers17030477

Figure Lengend Snippet: Figure 4. Effects of NO stress on COX-2 expression and PGE2 levels in human melanoma. NO stress significantly induced COX-2 expression (a) and PGE2 production (b) in melanoma cells. Full- length blots of manuscript is shown in Figures S7 and S8. A375 cells were cultured in serum-free DMEM medium with DetaNONOate 100 µM for 24 and 48 h. After 48 h, the media was collected for analysis of PGE2 levels using LC-MS/MS. The internal standard PGE2-d4 chromatogram is shown in Supplementary Figure S1. Cotreatment with nNOS inhibitor HH044 effectively diminished the induction of COX-2 (c) and PGE2 production (d) by IFN-γ in SK-MEL-28 and A375 cells, respectively. DMEM media was collected after treatment with HH044 20 µM and IFN-γ 250 units/mL for 48 h. The corresponding PGE2-d4 chromatogram is shown in Supplementary Figure S2. The impact of different treatments on PGE2 levels in human melanoma SK-MEL-28 cells is shown in Supplementary Figure S11. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—not significant.

Article Snippet: Mouse monoclonal anti-human nNOS (#MAB2416; Novus Biologicals, Centennial, CO, USA), mouse monoclonal anti-human β-Actin (#8H10D10; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-human PD-L1 (#E1L3N; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-human COX-2 (#D5H5; Cell Signaling Technology, Danvers, MA, USA) antibodies were used as primary antibodies, and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit (1:5000; 1:2000; Cell Signaling Technology, Danvers, MA, USA) were utilized as secondary antibodies for western blotting analysis.

Techniques: Expressing, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Cancers

Article Title: Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression.

doi: 10.3390/cancers17030477

Figure Lengend Snippet: Figure 6. Celecoxib enhances the cytotoxicity of nNOS inhibitor HH044 in melanoma cells. IC50 was determined using GraphPad Prism, detected by MTT colorimetric analysis. A375 cells were treated with various concentrations of HH044 and celecoxib for 72 h, and viable cells were measured by absorbance at 595 nm. All IC50 values are included in Supplementary Table S3.

Article Snippet: Mouse monoclonal anti-human nNOS (#MAB2416; Novus Biologicals, Centennial, CO, USA), mouse monoclonal anti-human β-Actin (#8H10D10; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-human PD-L1 (#E1L3N; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-human COX-2 (#D5H5; Cell Signaling Technology, Danvers, MA, USA) antibodies were used as primary antibodies, and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit (1:5000; 1:2000; Cell Signaling Technology, Danvers, MA, USA) were utilized as secondary antibodies for western blotting analysis.

Techniques:

Journal: Cancers

Article Title: Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression.

doi: 10.3390/cancers17030477

Figure Lengend Snippet: Figure 7. The nNOS inhibitor HH044 decreased tumor PGE2 levels in vivo. DBA/2 male mice were injected with Cloudman S91 cells to induce tumor growth. Mice were then randomized into different groups (vehicle control, HH044 10 mg/kg i.p., and celecoxib 50 mg/kg p.o. daily for 24 days). At the end of the study, tumors were collected and processed for PGE2 analysis using LC/MS-MS. * p < 0.05, ** p < 0.01, compared to control.

Article Snippet: Mouse monoclonal anti-human nNOS (#MAB2416; Novus Biologicals, Centennial, CO, USA), mouse monoclonal anti-human β-Actin (#8H10D10; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-human PD-L1 (#E1L3N; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-human COX-2 (#D5H5; Cell Signaling Technology, Danvers, MA, USA) antibodies were used as primary antibodies, and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit (1:5000; 1:2000; Cell Signaling Technology, Danvers, MA, USA) were utilized as secondary antibodies for western blotting analysis.

Techniques: In Vivo, Injection, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Cancers

Article Title: Crosstalk Between nNOS/NO and COX-2 Enhances Interferon-Gamma-Stimulated Melanoma Progression.

doi: 10.3390/cancers17030477

Figure Lengend Snippet: Figure 9. A schematic representation of the crosstalk between the nNOS/NO and COX-2/PGE2 signaling pathways, which enhanced IFN-γ-induced PD-L1 expression in melanoma. IFN-γ has been implicated as a pro-tumorigenic cytokine attributed to melanoma progression. Our study has demonstrated that the nNOS/NO and COX-2/PGE2 pathways are activated in the presence of IFN-γ, both of which generate proinflammatory molecules. The enzymatic product of COX-2 activity, PGE2, induced the expression of nNOS in melanoma cells, while NO produced by nNOS further increased the expression of COX-2. As a result, this feedforward loop amplifies the pro-tumorigenic effects of IFN-γ in melanoma by inducing PD-L1, leading to immune suppression within the tumor microenvironment.

Article Snippet: Mouse monoclonal anti-human nNOS (#MAB2416; Novus Biologicals, Centennial, CO, USA), mouse monoclonal anti-human β-Actin (#8H10D10; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-human PD-L1 (#E1L3N; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-human COX-2 (#D5H5; Cell Signaling Technology, Danvers, MA, USA) antibodies were used as primary antibodies, and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit (1:5000; 1:2000; Cell Signaling Technology, Danvers, MA, USA) were utilized as secondary antibodies for western blotting analysis.

Techniques: Protein-Protein interactions, Expressing, Activity Assay, Produced

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 2. Attenuation of mTOR prevents neurovascular uncoupling in 12-month-old hAPP(J20) mice. A, L-NPA at 200 nM has no impact on endothelium-dependent, eNOS-mediated, ACh- induced vasoreactivity. Pretreatment with L-NPA, a selective nNOS inhibitor, at 200 nM (close to its Ki for nNOS) has no impact on endothelium-dependent, eNOS-mediated vasoreactivity induced by superfusion of 10 mM ACh (Tukey’s test, q(8) = 0.94, p = 0.79 vs aCSF vehicle). In contrast, superfusion with 200 nM L-NPA together with 10 mM L-NAME, a general NOS inhibitor, profoundly inhibited ACh-induced CBF increases (91.7% inhibition, q(8) = 13.64, ppppp , 0.0001). B–D, NVC responses in hAPP mice; the fold change in CBF during whisker pad stimulation (30 s; bold black line) was measured sequentially in the presence of aCSF (vehicle; B), 200 nM L-NPA to inhibit nNOS (Fig. 2A; C), and 200 nM L-NPA plus 10 mM L-NAME (D) to inhibit all remain- ing NOS activity (i.e., eNOS). E, NVC responses during 30 s whisker stimulations. Area under the curve was calculated as an increase relative to baseline (i.e., only upward peaks). Baseline NVC is impaired in hAPP(J20) mice compared with WT littermates (q(27) = 10.41, ppppp , 0.0001). These deficits were negated by 8 months of rapamycin (Rapa; q(27) = 22.02, ppppp , 0.0001) and enhanced compared with WT (q(27) = 11.91, 1111p , 0.0001). Inhibition of nNOS with 200 nM L-NPA significantly reduced NVC in WT (q(27) = 5.24 vs baseline, pp = 0.02), but not in hAPP(J20) mice (q(27) = 0.38, p = 0.99 vs baseline), suggesting a preexisting nNOS deficit in hAPP(J20) animals that was negated by rapamycin treatment (q(27) = 7.14 vs baseline, pppp = 0.0008). The remaining NVC response in the presence of L-NPA plus L-NAME (inhibiting all remaining NOS activity; i.e., eNOS) was not significantly decreased with respect to L-NPA treatment in WT mice (q(27) = 2.31, p = 0.78) or in hAPP(J20) mice (q(27) = 0.76, p = 0.99) but was enhanced in rapamycin-treated hAPP(J20) mice (L-NPA1L-NAME vs

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Inhibition, Whisker Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 3. Short-term mTOR attenuation reverses NVC impairments in 12-month-old hAPP(J20) mice. A–C, Fold change in cerebral blood flow are the mean 6 SEM of n = 4–5/group during whisker pad stimulation (30 s, bold black line), stimulations conducted sequentially in the presence of aCSF [vehicle (Veh); A], 200 nM L-NPA to inhibit nNOS specifically (Fig. 2A; B), and 200 nM L-NPA110 mM L-NAME (C) to inhibit all NOS, thus defining the contribution of the non-nNOS-dependent, L-NAME-sensitive eNOS. D, NVC responses during 30 s whisker stimulations. Area under the curve was calculated as an increase relative to baseline (i.e., only upward peaks). Baseline NVC impairments in hAPP(J20) mice relative to WT (q(33) = 12.81, ppppp , 0.0001) are reversed by 2 months of rapamycin (Rapa; q(33) = 26.01, ppppp , 0.0001), with enhancement of NVC in rapamycin-treated hAPP(J20) mice compared with WT (q(33) = 13.93, ppppp , 0.0001). L-NPA superfusion significantly reduces NVC in WT mice (q(33) = 5.25, pp = 0.02), but not in hAPP(J20) mice (q(33) = 0.22, p . 0.99), suggesting a preexisting nNOS deficit in this group that is restored by rapamycin treatment in the hAPP(J20)1rapamycin group, indicated by a significant inhibition of NVC in the presence of 200 nM L-NPA (q(33) = 12.28 vs base- line, ppppp , 0.0001). The remaining NVC response in the presence of L-NPA1L-NAME (inhibiting all remaining NOS activity; i.e., eNOS) was not significantly different versus L-NPA alone in WT (q(33) = 1.05, p = 0.99), hAPP(J20) (q(33) = 0.09, p . 0.99), or in hAPP(J20)1rapamycin (q(33) = 3.60, p = 0.24). E, Contributions of nNOS, eNOS, and non-NOS components to total NVC. Deficits in nNOS-dependent NVC in hAPP(J20) mice (q(33) = 4.47, ppp = 0.009 vs WT) are negated by mTOR inhibition in rapamycin-treated hAPP(J20) mice (q(33) = 11.31, ppppp , 0.0001 vs hAPP) and nNOS-dependent NVC is enhanced over WT (q(33) = 7.10, 1111p , 0.0001). Additionally, rapamycin enhances L-NAME-sensitive activities (eNOS) in hAPP(J20) mice relative to vehicle-treated hAPP(J20) mice (q(33) = 3.93, pp = 0.02). Deficits in the remaining non-NO-mediated NVC response in hAPP(J20) mice (q(33) = 6.06, pppp = 0.0004 vs WT) are negated in rapamycin-treated hAPP(J20) mice [q(33) = 7.87, ppppp , 0.0001 vs vehicle-treated hAPP(J20) mice]. F, Hippocampal-dependent contextual memory impairment in 12-month-old hAPP(J20) mice [hAPP(J20) vs WT, q(16) = 4.19, pp = 0.024] is negated by 2 months of rapamycin treatment [q(16) = 3.93, pp = 0.034, hAPP(J20) vs hAPP(J20)1rapamycin]. G, Baseline NVC response is correlated with contextual memory performance (r = 0.668, p = 0.009). Behavioral studies used n = 6–7 mice/group. All post hoc analyses are Tukey’s multiple-comparisons tests. Unless other- wise indicated, asterisks (p) in the figure represent a significant difference relative to hAPP1Veh and pluses (1) represent a significant difference relative to WT1Veh.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Whisker Assay, Inhibition, Activity Assay

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 4. mTOR-driven NVC deficits precede contextual memory impairment in 6-month-old hAPP(J20) mice. A, Hippocampal-dependent contextual memory is intact in 6-month-old hAPP (J20) mice (F(2,16) = 0.33, p = 0.73, no change). B–D, Fold change in cerebral blood flow from whisker pad stimulation (30 s, bold black line, representing mean 6 SEM of n = 5/group) was measured sequentially in the presence of aCSF [vehicle (Veh); B], 200 nM L-NPA to selectively inhibit nNOS (Fig. 2A; C), and 200 nM L-NPA110 mM L-NAME to inhibit all remaining NOS activity (i.e., eNOS; D). E, Total NVC responses during 30 s whisker stimulations. Area under the curve was calculated as an increase relative to baseline (i.e., only upward peaks). Baseline NVC impair- ment in 6-month-old hAPP(J20) mice versus WT (q(35) = 8.71, 1111p , 0.0001) is reversed by rapamycin (Rapa) treatment (2months, q(35) = 21.07, ppppp , 0.0001) and NVC is enhanced in hAPP(J20)1rapamycin compared with WT (q(35) = 12.36, 1111p , 0.0001). A significant reduction in NVC by L-NPA superfusion (q(35) = 11.75, ppppp , 0.0001) in the hAPP(J20)1rapamycin group, which is absent in vehicle-treated hAPP(J20) mice (q(35) = 0.26, p . 0.99), indicates that rapamycin restores nNOS-dependent NVC in hAPP(J20) mice. L- NPA1L-NAME-sensitive (i.e., eNOS-mediated) NVC was not significantly reduced in WT (q(35) = 3.40, p = 0.32), hAPP(J20) (q(35) = 0.87, p = 0.99), or hAPP(J20)1rapamycin groups (q(35) = 3.04, p = 0.46) relative to L-NPA alone. F, Contributions of nNOS, eNOS, and non-NOS components to total NVC. Impaired L-NPA-sensitive, nNOS-driven NVC responses in hAPP(J20) mice (q(35) = 3.67, pp = 0.036) are negated by rapamycin (2months, q(35) = 11.28, ppppp , 0.0001), and the nNOS-driven NVC response is enhanced versus WT groups (q(35) = 7.60, 1111p , 0.0001). eNOS-mediated, L-NPA1L-NAME-sensitive NVC is unchanged at 6 months of age in all groups; mTOR attenuation enhances non-NOS-dependent NVC in rapamycin-treated hAPP(J20) mice [q(35) = 4.27, 1p = 0.013 vs WT mice; q(35) = 7.09, ppppp , 0.0001 vs hAPP(J20) mice]. G, Progressive, early-onset NVC impairments in hAPP(J20) mice modeling AD. NVC impairments in hAPP(J20) mice are present at 6 months (q(23) = 6.18, 11p = 0.003) and are significantly exacerbated by 12 months of age (q(23) = 5.55, ppp = 0.008). During this same time period, NVC remains unchanged in both WT animals (q(23) = 1.17, p = 0.96, not significant) and in hAPP(J20) mice treated with rapamycin (q(23) = 2.10, p = 0.67, not significant). All post hoc tests are Tukey’s multiple-comparisons tests. N = 5 mice/group except for behavioral studies (n = 6–7/group). Unless otherwise indicated, asterisks (p) in the figure represent a significant dif- ference relative to hAPP1Veh and pluses (1) represent a significant difference relative to WT1Veh.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Whisker Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 5. mTOR decreases nNOS abundance and inhibits nNOS phosphorylation at its primary activation site in cultured neuronal cells. A, Representative electropherograms of chemilumines- cent signals detected by capillary Western immunoassay (Wes; Beekman et al., 2018) measuring total nNOS from hippocampal formation of patients at Braak stages III–VI compared with non- AD controls (C). B, Quantitative analysis shows decreased nNOS abundance in the hippocampal formation with progression of Braak pathology (Spearman’s r = 0.975, p = 0.02). Data are a scatter plot overlaid with the regression line and 95% CI representing n = 3/group. C, Representative electropherograms of chemiluminescent signals detected by capillary Wes measuring total nNOS (152 kDa), eNOS (140 kDa), and b -actin (48 kDa) in isolated microvasculature from hAPP(J20) mice at 6 or 12months (mo.) of age that received 2 months of vehicle (Veh) or rapamycin (Rapa)-containing diets. D, Total nNOS is reduced in 6-month-old hAPP(J20) mice modeling early-stage preclinical AD (Sidak’s test, t(17) = 5.42, p = 0.0001). Two months of mTOR attenuation restored nNOS expression in hAPP(J20) mice treated with rapamycin (Sidak’s test, t(17) = 7.37, p , 0.0001). No change in total nNOS was observed in 12-month-old hAPP(J20) mice. E, Total eNOS protein expression is unchanged by age (F(2,18) = 0.005, p = 0.94), treatment (F(2,18) = 1.19, p = 0.33), or their interaction (F(2,18) = 2.67, p = 0.10). F, Representative electropherograms of chemiluminescent signals detected by capillary Wes in lysates from N2a cells. G–J, Quantitative analyses of data in F indicate that rapamycin (5.5 nM) treatment inhibits mTOR in N2a cells (81.7% reduction in S6 phosphorylation, t(10) = 23.5, ppppp , 0.0001; G); increases nNOS Ser1412 phosphorylation (t(10) = 31.2, ppppp , 0.0001; H) and total nNOS levels (t(14) = 9.3, ppppp , 0.0001; I); and increases Hsp90 levels (t(10) = 4.0, ppp = 0.003) in N2a cells (J). Data are scatter plots for n = 8 controls and n = 4 rapamycin-treated N2a cultures, from n = 4 inde- pendent experiments performed on different days, overlaid on a bar graph representing mean 6 SEM.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Phospho-proteomics, Activation Assay, Cell Culture, Western Blot, Isolation, Expressing

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 7. mTOR-dependent regulation of cerebrovascular function through nitric oxide synthases. mTOR inhibits neurovascular coupling through the regulation of nNOS-, eNOS-, and non- NO-dependent components of the neurovascular coupling response. Our prior studies indicate that mTOR drives cerebrovascular reactivity deficits and reduced baseline cerebral blood flow through the inhibition of eNOS activity (Van Skike and Galvan, 2018) in models of AD ( Lin et al., 2013, 2017; Van Skike et al., 2018), in models of vascular cognitive impairment (Jahrling et al., 2018; Van Skike et al., 2018), and in normative aging (Van Skike et al., 2020). Figure created in part with BioRender.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Inhibition, Activity Assay

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure4. nanos1mRNAisaSmaug2targetinembryoniccorticalprecursors.A,RT-PCRfornanos1,nanos2,andnanos3mRNAsinmurinecorticesfromE11tobirth(P0).nanos1mRNAexpression wasdetectedusingtwodifferentprimersets.PCRproductsweresequencedtoconfirmspecificity.ve,SamplewithknownexpressionoftargetmRNAandusedasapositivecontrolforthereaction; -ve,samplegeneratedintheabsenceofreversetranscriptase.B,SchematicofSREsinthenanos1mRNAtranscript.YellowarrowlabeledCDSrepresentstheprotein-codingregion.C,Westernblot analysisforNanos1inE11.5to2-month-oldcortices.TheblotwasreprobedforERK1/2asaloadingcontrol.D,WesternblotofHEK-293TcellstransfectedwithaFlag-taggedmouseSmaug2construct andimmunoprecipitatedwithanti-Smaug2orwithcontrolnonspecificrabbitIgG,probedwithantibodiesforSmaug2.Asacontrol,10%oftheinputhomogenatewasloaded.E,Westernblot(top) of E12.5 cortical lysates immunoprecipitated with the same Smaug2 antibody as in D or with control, nonspecific rabbit IgG and probed with anti-Smaug2. As a positive control, 10% of the input homogenatewasloaded.Similarimmunoprecipitatesweregeneratedinparallel,mRNAwasextracted,andthesampleswereanalyzedfornanos1,nanos2,andnanos3mRNAsusingRT-PCR(second tobottompanels).F,ConfocalimagesofFISHfornanos1(left),nanos2(center),andnanos3(right)mRNAs(blackgranules)incoronalsectionsoftheE12.5cortex.v,Ventricle.Scalebar,10m.G, Higher-magnificationconfocalimagesoftheVZ/SVZofanE13.5corticalsectionshowingFISHfornanos1mRNA(red)andimmunostainingforSmaug2(green).Top,Merge.Boxedregionsareshown at higher magnification in the right panels, which also show colocalization of Smaug2 and nanos1 mRNA on the z-axis (XZ and YZ), as indicated by the hatched (Figure legend continues.)

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: Immunoprecipitation, Control, Positive Control

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure 5. Nanos1 is necessary and sufficient to promote neurogenesis in vivo. A, Western blots of HEK-293T cell lysates cotransfected with murine Nanos1 or Flag-tagged murine Nanos2 or Nanos3 expression constructs and a control shRNA (Con) or a Nanos1 shRNA (shNos1) and probed with anti-Nanos1 or anti-Flag, as indicated. The blots were reprobed with ERK1/2 as a loading control. B–H, E13/E14 murine cortices were coelectroporated with a nuclear EGFP construct, and either a control (con) or Nanos1 shRNA (shNos1) and coronal sections were analyzed 3 d later at E16/E17.B,ImagesofelectroporatedsectionsimmunostainedforEGFP(green).v,Ventricle.Scalebar,10m.C,QuantificationofsectionssimilartothoseinBforthepercentageofEGFP-positive cells located in the different cortical regions. **p 0.01. n 3 embryos each, at least 3 sections per embryo. D, Confocal micrographs of the VZ/SVZ (three top rows) or CP (bottom row) of electroporatedsectionsimmunostainedforEGFP(green)andPax6,Ki67,Tbr2,orSatb2(allred).Arrowsindicatedouble-labeledcells.v,Ventricle.Scalebar,10m.E–H,Quantificationofsections similartothoseinDforthepercentageofEGFP-positivecellsthatexpressedPax6(E),Ki67(F),Tbr2(G),orSatb2(H).**p0.01.***p0.001.n3embryoseach,atleast3sectionsperembryo. I–K,E13/E14corticeswerecoelectroporatedwithanuclearEGFPconstructandacontrol(con)orNanos1shRNA(shNos1) anshRNA-resistanthumanNanos1expressionvector(resc)andcoronal sections were analyzed 3 d later at E16/E17. I, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 m. J, K, Sections similar to those in I were immunostained for EGFP and Pax6 or Satb2 and the proportion of EGFP-positive cells that were also positive for the marker was quantified. **p 0.01. (Figure legend continues.)

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: In Vivo, Western Blot, Expressing, Construct, Control, shRNA, Marker

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure6. Smaug2andnanos1mRNAareassociatedwith4E-TinaP-Body-likegranuleinPax6-positiveapicalprecursors.A,WesternblotanalysisforSmaug2(Smg2)and4E-TinlysatesofE12.5 corticalprecursorsculturedfor3dandimmunoprecipitatedwithanti-Smaug2orwithcontrol,nonspecificrabbitIgG.Asapositivecontrol,10%oftheinputhomogenatewasloaded.B,Westernblot analysisforSmaug2and4E-TinlysatesofE12.5corticalprecursorsculturedfor3dandimmunoprecipitatedwithanti-4E-Torwithcontrol,nonspecificmouseIgG.Asapositivecontrol,10%ofthe inputhomogenatewasloaded.C,ConfocalimagesofE12.5corticalprecursorsculturedfor3dandimmunostainedforSmaug2(green)and4E-T(magenta).Cultureswerealsocounterstainedwith Hoechst(blue).Top,Boxedareasareshownathighermagnificationinthebottompanels.ArrowsindicategranulesthatarepositiveforbothSmaug2and4E-T.Scalebar,5m.D,Confocalimages ofE12.53dcorticalprecursorculturesafterthePLAwithSmaug2and4E-Tantibodies.CultureswerealsocounterstainedwithHoechst(blue).Left,Boxedareasareshownathighermagnification totheright.Scalebar,10m.E,ConfocalimagesofE12.5corticalprecursorsculturedfor3dandimmunostainedforSmaug2(red)andDcp1(green).CultureswerealsocounterstainedwithHoechst (blue). Top, Boxed areas are shown at higher magnification in the bottom panels. Arrows indicate granules that are double labeled for Smaug2 and Dcp1. Scale bar, 10 m. F, Confocal images of cortical precursor cultures after PLA with Smaug2 and Dcp1 antibodies. Cultures were also counterstained with Hoechst (blue). Left, Boxed areas are shown at higher magnification on the right. Scale bar, 10 m. G, RT-PCR analysis for nanos1 mRNA in 4E-T immunoprecipitates (4E-T IP) from the E12.5 cortex. As a control, similar lysates were (Figure legend continues.)

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Control

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure7. KnockdownofSmaug2or4E-TcausesaberrantNanos1expression,andthisisresponsiblefortheSmaug2knockdown-mediatedincreaseinneurogenesis.A,Nanos1immunoreactivity inacoronalsectionoftheE16/E17cortex.v,Ventricle.Scalebar,10m.B,ConfocalimagesofcellsattheborderoftheSVZandtheIZofE16/E17murinecorticesthatwerecoelectroporated3dearlier withanuclearEGFPconstructandcontrol(con)orSmaug2(shSmg2)shRNAs.SectionswereimmunostainedforEGFP(green)andNanos1(red).ArrowsandarrowheadsindicateEGFP-positivecells that do or do not express Nanos1, respectively. Scale bar, 10 m. C, Quantification of the proportion of EGFP-positive cells expressing detectable Nanos1 in sections similar to those in B. ***p 0.001. n 3 embryos each, at least 3 sections per embryo. D, Confocal images of cells at the border of the SVZ and the IZ of E15/E16 murine cortices that were coelectroporated 2 d earlier with a nuclearEGFPconstructandcontrol(con)or4E-T(sh4ET)shRNAs.SectionswereimmunostainedforEGFP(green)andNanos1(red).ArrowsandarrowheadsindicateEGFP-positivecellsthatdoordo notexpressNanos1,respectively.Scalebar,10m.E,QuantificationoftheproportionofEGFP-positivecellsexpressingdetectableNanos1insectionssimilartothoseinD.*p0.05.n3embryos each,atleast3sectionsperembryo.F–I,E13/E14corticeswerecoelectroporatedwithanuclearEGFPconstructandcontrol(con)orSmaug2shRNA(shSmg2) Nanos1shRNA(shNos1),andcoronal corticalsectionswereanalyzed3dlateratE16/E17.F,ImagesofelectroporatedsectionsimmunostainedforEGFP(green).v,Ventricle.Scalebar,10m.G,Quantificationofsectionssimilartothose in F for the percentage of EGFP-positive cells located in the different cortical regions. *p 0.05. **p 0.01. ns, Nonsignificant. n 3 embryos each, at least 3 sections per embryo. H, I, Quantification of EGFP-positive, marker-positive cells in sections as in F immunostained for EGFP and either Pax6 (H) or Satb2 (I). *p 0.05. **p 0.01. n 3 embryos each, at least 3 sections per embryo. J, Schematic showing the proposed repressive complex involving Smg2, 4E-T, Dcp1, and nanos1 mRNA (top). When the complex is disrupted, either by environmental signals or by knockdownofcomplexcomponentssuchasSmaug2,thiscausesaberranttranslationofNanos1,therebypromotingneurogenesis(bottom).G–I,StatisticswereperformedwithANOVAandTukey’s post hoc multiple comparisons test. Other panels, Statistics were performed with Student’s t test. Error bars indicate SEM.

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: Expressing, Marker