Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 1. PPX induces autophagy through a DRD3-dependent mechanism in mouse striata and HEK cells. (A) Western blot and densitometric analysis for LC3 and p62 in WT and drd3KO mice treated with 0.15 mg/kg/d for six days (n = 5). PPX promotes a decline in LC3-II and p62 in WT but not in drd3KO mice. (B–D) Autophagic flux in HEK (n = 7–8; (B)), DRD2-HEK (n = 10; (C)), and DRD3-HEK (n = 8–19; (D)) cells treated with the autophagosome–lysosome fusion blocker chloroquine (CQ, 20 µM, 1 h; lanes 3 and 4) and 0.1 µM PPX (lanes 5 and 6) or 10 µM PPX (lanes 7 and 8) for 4 h. CQ was added after PPX. Western blot for LC3 revealed that autophagic flux (LC3-II levels in CQ + PPX vs. CQ) was increased after PPX treatment in DRD3-HEK cells (D) but not in HEK (B) or DRD2-HEK cells (C). Statistical analyses were performed using the unpaired t-test for mice and Mann–Whitney test for cells. n = number of mice per experimental group or number of experimental repeats in cells; ns = non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Western Blot, MANN-WHITNEY

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 3. DRD3-induced autophagy is prolonged in cells expressing a pathogenic form of polyQ-HTT. The effects of PPX (0.1 µM) on LC3-II levels were analyzed in the absence (lanes 3 and 4) and presence (lanes 7 and 8) of the autophagosome–lysosome fusion blocker chloroquine (CQ, 20 µM, 1 h; lanes 5 and 6). (A–C) After 4 h of PPX treatment (n = 5–9), the autophagic flux (LC3-II levels in CQ + PPX vs. CQ) was increased in DRD3- (A), Q23-DRD3- (B), and Q74-DRD3-HEK (C) cells. (D–F) After 24 h of PPX treatment (n = 6), the autophagic flux remained increased in Q74-DRD3-HEK cells (D) but not in DRD3-HEK (E) or Q23-DRD3-HEK cells (F). Statistical analyses were performed using the Mann–Whitney test. n = number of experimental repeats; ns = non-significant; * p < 0.05.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Expressing, MANN-WHITNEY

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 5. PPX promotes Q74 clearance and protects DRD3-HEK cells against its genotoxic effect. (A) Western blot for polyQ in Q74-DRD3- (n = 12), Q74- (n = 7), and Q23-DRD3-HEK (n = 7) cells treated with PPX (6 h within 12 h after transfection). The densitometric analysis showed that PPX promotes a significant decrease in Q74 but not Q23 in DRD3-HEK cells, nor Q74 in HEK cells (Kruskal– Wallis followed by Dunn’s multiple comparison test). (B–E) Fluorescent labeling and quantitative analysis of the number of pyknotic nuclei (B,D) and immunofluorescent intensity of polyQ- and γH2AX expression in Q74-DRD3- and Q23-DRD3-HEK cells treated with PPX (C,E). PPX reduced the number of pyknotic nuclei (B(iv,v),D) and polyQ and γH2AX immunofluorescent intensity (C(iv,v),E) in Q74-DRD3-HEK cells without affecting Q23-DRD3-HEK cells. Statistical analyses were performed using ANOVA followed by Bonferroni’s (number of pyknotic nuclei and H2AXγ labeling intensity) and Brown–Forsythe tests, followed by Sidak’s (polyQ labeling intensity) multiple comparison tests. Arrows in (B) indicate pyknotic nuclei. A.U., arbitrary units. Bar: in (B(v)) (for (B(i–v))): 50 µm; in (C(v)) (for (C(i–v))): 10 µm. n = number of experimental repeats; *** p < 0.001. ns, not significant.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Western Blot, Transfection, Comparison, Labeling, Expressing

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 6. Short treatment with PPX (0.15 mg/kg, 6 days) induces mTORC1 inhibition through a DRD3- dependent mechanism. Western blot and densitometric analysis for the total and phosphorylated forms of p70S6K at Thr389 (A), AMPKα at Thr172 (B), and ULK1 at Ser at 757 and Ser555 (C) in striatal extracts of WT and drd3KO mice. PPX promoted a decrease in Thr389-p70S6K and Ser757-ULK phosphorylation in WT mice without affecting Thr172-AMPKα and Ser555-ULK phosphorylation. No phosphorylation changes were detected in drd3KO mice. n = 5–9 mice per experimental group, unpaired t-test; ns = non-significant; * p < 0.05; ** p < 0.01.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Inhibition, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 8. DRD3 promotes Q74 clearance through AMPK. Western blot and densitometric analysis of the total and phosphorylated forms of AMPKα at Thr172 (A) and Q74 in Q74-DRD3-HEK cells (B). PPX (0.1 µM, 6 h) induces Thr172-AMPKα phosphorylation ((A), lanes 3 and 4), rescues Thr172- AMPKα dephosphorylation induced by the AMPK inhibitor compound C (CC; 2 µM, 6 h; (A), lanes 7 and 8), and reduces Q74 levels in Q74-DRD3-HEK cells ((B), lanes 4 and 5) and Q74-DRD3-HEK cells treated with CC ((B), lanes 8 and 9). Lane 1 in (B), polyQ-untransfected DRD3-HEK cells. n = 6–8 experimental repeats. Statistical analyses were performed using ANOVA followed by the Fisher LSD test in the study of AMPKα phosphorylation and ANOVA followed by the Holm–Sidak multicomparison test in the study of Q74 levels. * p < 0.05. Analysis of S2488-mTOR was inconclusive since no differences among treatments resulted from either mTOR-independent AMPK activation or the recovery of transient mTOR inhibition in non-Q74 transfected cells.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Western Blot, Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Inhibition, Transfection

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 10. Schematic view of signaling pathways involved in transient and persistent DRD3-medited autophagy in WT and R6/1 mice, respectively. In WT mice (left), autophagy is transiently induced by mTORC1 inhibition with the subsequent inhibition of p70S6K and the activation of ULK1. In R6/1 mice (right), where mTORC1 is hyperphosphorylated, autophagy is persistently activated through AMPK with the direct activation of ULK1, promoting an effective clearance of soluble mHTT. In persistent autophagy, the mTORC1-p70S6K branch of mTORC1 signaling is also inhibited, but mTORC1-ULK1 signaling is not activated. In addition, the crosstalk between mTORC1-p70S6K and MAPK-p90S6K pathways is activated and rpS6 activity is preserved. Blue arrows and blocked red lines indicate the activation or inhibition of downstream targets, respectively. Dashed red lines indicate putative pathways of mTORC1 inhibition in persistent autophagy.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Protein-Protein interactions, Inhibition, Activation Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin phosphorylation in vitro and after hypoxia. A, primary amino acid sequence of neuroglobin showing putative phosphorylation sites and binding sites for 14-3-3. B, in vitro phosphorylation of recombinant neuroglobin (Ngb) using purified ERK1 and ERK2 with and without 10 μm U0126 (inhibitor). C, in vitro phosphorylation of recombinant neuroglobin using purified recombinant PKA in the presence and absence of the PKA synthetic peptide inhibitor. D, quantitative analysis of neuroglobin phosphorylation with and without specific respective inhibitors using NIH Image software. E, immunoblot analysis of wild-type and mutant neuroglobin expression in SH-SY5Y neuronal cells that overexpress neuroglobin. F, increased ERK1/2 activity after 3 h of hypoxia as determined by immunoblot analysis using anti-phospho-ERK (pERK) antibody (upper panel). Total ERK1/2 protein was unchanged during hypoxia (middle panel). G, neuroglobin-GFP-expressing cells were subjected to either normoxia or 1% hypoxia for 3 h, metabolically labeled with inorganic orthophosphate, and immunoprecipitated (IP) with anti-neuroglobin antibody. The immunoprecipitated proteins were resolved by SDS-PAGE, and the phosphorylation signals were detected by autoradiogram. Neuroglobin phosphorylation was significantly increased in neuroglobin-GFP-expressing neuronal cells in vivo after 3 h of hypoxia compared with normoxia. GSD, glucose- and serum-deprived medium. H, quantitative analysis of in vivo neuroglobin phosphorylation using NIH Image software.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Phospho-proteomics, In Vitro, Sequencing, Binding Assay, Recombinant, Purification, Software, Western Blot, Mutagenesis, Expressing, Activity Assay, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: ERK inhibitor FR180204 inhibits neuroglobin phosphorylation in neuronal cells after hypoxia. A, neuroglobin-expressing cells were cultured and subjected to normoxic and hypoxic conditions in the presence and absence of 0.5 μm FR180204 for 3 h. After 3 h, the cells were lysed, and immunoblotting was performed with anti-phosphoserine antibody (αPS). Ngb, neuroglobin. B, the same blot was stripped and probed with anti-β-actin antibody.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin association with 14-3-3 under hypoxia. A and B, GFP- and neuroglobin (Ngb)-expressing cells were subjected to either normoxia or 1% hypoxia, lysed, and immunoprecipitated (IP) with anti-14-3-3 antibody, followed by probing with anti-pan-14-3-3 and anti-GFP antibodies. C, quantitative analysis revealed that neuroglobin binding to 14-3-3 was increased in neuronal cells during hypoxia. D and E, time-dependent increase in 14-3-3 binding to neuroglobin after hypoxia. Neuroglobin-GFP-expressing cells were subjected to either normoxia or 1% hypoxia at the indicated times, lysed, immunoprecipitated with anti-neuroglobin antibody, and immunoblotted with anti-GFP and anti-14-3-3 antibodies, respectively. F, quantitation of the time-dependent increase in 14-3-3 binding to neuroglobin. G, neuroglobin association with 14-3-3 in intact live cells as determined by FRET analysis. Cells were exposed to normoxia or hypoxia for 3 h prior to analysis for protein-protein interaction by FRET. Cells were either singly transfected (upper panels) or cotransfected with YFP-14-3-3 and CFP-neuroglobin (Neu; middle and lower panels). The panels show CFP, YFP, merge, or FRET (pseudo-color, far right panels); the latter represents intensity of the FRET signal.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Quantitation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin association with 14-3-3 as studied by FRET. Shown in the upper panels are single cell imaging showing cell fluorescence before and after photobleaching for cells transfected with CFP and YFP (A), cells transfected with WT CFP-neuroglobin and YFP-14-3-3 under normoxic conditions (B), cells transfected with WT CFP-neuroglobin and YFP-14-3-3 under hypoxic conditions (C), cells transfected with CFP-S17A neuroglobin and YFP-14-3-3 under hypoxic conditions (D), and cells transfected with CFP-S50A neuroglobin and YFP-14-3-3 under hypoxic conditions (E). In the lower panels of A–E are shown the fluorescence signals from CFP (green traces) and YFP (red traces) measured along the region marked with the red arrows (x axis indicates the progress of the fluorescence detector along the red arrow). When CFP and YFP are not close (and neuroglobin and 14-3-3 are therefore not close), photobleaching is expected to decrease the YFP signal (red) with very little effect on the CFP signal (green). Alternatively, when CFP and YFP are close enough (and neuroglobin and 14-3-3 are therefore close), photobleaching will decrease the YFP signal (red) and increase the CFP signal (green) because CFP cannot transfer energy to YFP anymore (see “Results” for details). The increases in green fluorescence accompanied by larger decreases in red fluorescence are marked in the C and D, indicating protein interaction between 14-3-3 and neuroglobin after hypoxia occurred in WT and S17A neuroglobin. In A, B, and E, there is a decrease in red fluorescence, but the profile for green fluorescence is not changed after photobleaching, indicating poor FRET between CFP and YFP, suggesting very little interaction between 14-3-3 and neuroglobin. These results suggest that neuroglobin Ser50 plays a predominant role in 14-3-3 binding. F, the bar graph shows quantification of FRET efficiency under experimental conditions. FRET efficiency (percent) was calculated and graphed from n = three experiments and >12 randomly selected cells for each condition that was analyzed. S50A mutant neuroglobin showed a significantly reduced interaction with 14-3-3 compared with S17A mutant and wild-type neuroglobin.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Imaging, Fluorescence, Transfection, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Expression of endogenous neuroglobin and its association with 14-3-3 in brain. A, neuroglobin expression in different regions of sheep brain as assessed by immunoblotting with neuroglobin-specific polyclonal antibody. B and C, neuroglobin (Ngb) association with 14-3-3. Co-immunoprecipitations were performed using anti-neuroglobin and anti-14-3-3 antibodies from 100 μg of proteins from sheep fetal and adult brainstem and thalamus as described under “Experimental Procedures.” The immune complex was resolved on 4–12% NuPAGE gradient gel, transferred to nitrocellulose, and immunoblotted with anti-neuroglobin antibody (B) and anti-14-3-3 antibody (C).

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: 14-3-3 binding stabilizes neuroglobin phosphorylation. A, protein expression in 14-3-3ϵ, 14-3-3ζ, and control siRNA-silenced neuroglobin-GFP-expressing neuronal cells. mAb, monoclonal antibody. B, β-actin expression in 14-3-3 and control siRNA-silenced neuroglobin-GFP-expressing neuronal cells under the same conditions. C,14-3-3 protein expression is significantly reduced in 14-3-3 siRNA-treated cells as quantitated by NIH Image software. D, control and 14-3-3 siRNA-silenced neuronal cells were metabolically labeled with inorganic phosphate, immunoprecipitated with anti-neuroglobin antibody, and resolved by SDS-PAGE, and the signals were detected by autoradiogram. Shown is the phosphorylation of neuroglobin in 14-3-3 and control siRNA-treated cells in vivo after 3 h of hypoxia. E, quantitative analysis by NIH Image software reveals that neuroglobin (Ngb) phosphorylation is significantly reduced in 14-3-3 siRNA-treated cells.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Binding Assay, Phospho-proteomics, Expressing, Control, Software, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin phosphorylation increases nitrite reductase activity. A, phosphorylation of neuroglobin (Ngb) by ERK2 in vitro. B, bimolecular rate constants (BRC) calculated for the reaction of untreated or phosphorylated neuroglobin with nitrite. Phosphorylation increased nitrite reductase activity by 3-fold. C and D, spectral changes in non-phosphorylated (C) and phosphorylated (D) neuroglobin during the reaction with nitrite. E, absorbance traces indicate the qualitative change in the rates for the reaction of phosphorylated and non-phosphorylated neuroglobin with nitrite. F, phosphorylation increases five-coordination as indicated by faster rates of CO binding. Shown is the normalized decay of deoxyneuroglobin (due to the concomitant increase in neuroglobin-CO) after mixing phosphorylated/non-phosphorylated neuroglobin (5 μm) with half-saturated CO buffer ([CO] ≈ 500 mm). The inset shows representative reaction traces at 425 nm. Abs, absorbance.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Phospho-proteomics, Activity Assay, In Vitro, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Effect of 14-3-3 binding on nitrite reductase activity. A, non-phosphorylated and phosphorylated neuroglobin (Ngb) with ERK2. B, Coomassie Blue staining of non-phosphorylated and phosphorylated neuroglobin. C, 14-3-3-dependent nitrite reductase activity of phosphorylated and non-phosphorylated neuroglobin after purified 14-3-3 protein addition. BRC, bimolecular reaction rates.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Binding Assay, Activity Assay, Staining, Purification

Journal: Cells

Article Title: Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells

doi: 10.3390/cells10123394

Figure Lengend Snippet: Description of the quantitative changes related to the proteins belonging to the clusters identified by STRING analysis in NGB-overexpressing SH-SY5Y cells.

Article Snippet: The immunoprecipitates were checked by immunoblotting analysis, using anti-LC3-II mAb (Abcam), anti-LAMP1 mAb (Santa Cruz Biotechnology, Dallas, TX, USA) and for the detection NGB using anti-NGB pAb (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Membrane

Journal: Cells

Article Title: Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells

doi: 10.3390/cells10123394

Figure Lengend Snippet: Effect of NGB overexpression on autophagy induction in SH-SY5Y cells. ( A ) SH-SY5Y cells, SH-SY5Y cells transfected with an empty construct (CTRL) and stably transfected SH-SY5Y FLAG-NGB cells were lysed in lysis buffer, subjected to 15% SDS-PAGE and analyzed by Western blot using anti-LC3 polyclonal antibody or rabbit anti-SQSTM1 mAb. Loading control was evaluated using anti-ACTB mAb. A representative experiment among 3 is shown. ( B ) Bar graph on the right shows densitometric analysis. Results represent the mean ± SD from 3 independent experiments. *** p < 0.001, **** p < 0.0001.

Article Snippet: The immunoprecipitates were checked by immunoblotting analysis, using anti-LC3-II mAb (Abcam), anti-LAMP1 mAb (Santa Cruz Biotechnology, Dallas, TX, USA) and for the detection NGB using anti-NGB pAb (Santa Cruz Biotechnology).

Techniques: Over Expression, Transfection, Construct, Stable Transfection, Lysis, SDS Page, Western Blot, Control

Journal: Cells

Article Title: Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells

doi: 10.3390/cells10123394

Figure Lengend Snippet: Effect of NGB overexpression on LAMP1 expression in SH-SY5Y cells. ( A ) SH-SY5Y cells, SH-SY5Y cells transfected with an empty construct (CTRL) and stably transfected SH-SY5Y-NGB-FLAG cells were lysed in lysis buffer, subjected to 7.5% SDS-PAGE and analyzed by Western blot using anti-LAMP1 mAb. Loading control was evaluated using anti-ACTB mAb. A representative experiment among 3 is shown. ( B ) Bar graph shows densitometric analysis. Results represent the mean ± SD from 3 independent experiments. * p < 0.05, *** p < 0.001.

Article Snippet: The immunoprecipitates were checked by immunoblotting analysis, using anti-LC3-II mAb (Abcam), anti-LAMP1 mAb (Santa Cruz Biotechnology, Dallas, TX, USA) and for the detection NGB using anti-NGB pAb (Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing, Transfection, Construct, Stable Transfection, Lysis, SDS Page, Western Blot, Control

Journal: Cells

Article Title: Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells

doi: 10.3390/cells10123394

Figure Lengend Snippet: NGB associates with LC3-II during autolysosome formation in SH-SY5Y cells. ( A ) SH-SY5Y cells transfected with an empty construct (CTRL) and stably transfected SH-SY5Y-NGB-FLAG cells untreated or treated with HBSS for 2 h at 37 °C, were lysed in lysis buffer, followed by immunoprecipitation with rabbit anti-LC3-II. A rabbit IgG isotypic control (IpCtr) was employed. The immunoprecipitates were checked by Western blot analysis, using anti-LC3-II mAb, anti-LAMP1 mAb and anti-NGB pAb. A representative experiment among 3 is shown. ( B ) Bar graph shows densitometric analysis. Results represent the mean ± SD from 3 independent experiments. ** p < 0.005.

Article Snippet: The immunoprecipitates were checked by immunoblotting analysis, using anti-LC3-II mAb (Abcam), anti-LAMP1 mAb (Santa Cruz Biotechnology, Dallas, TX, USA) and for the detection NGB using anti-NGB pAb (Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Stable Transfection, Lysis, Immunoprecipitation, Control, Western Blot