nf kb subunit p65 antibody  (Proteintech)

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT , SOD1 G93A , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human SOD1 WT or SOD1 G93A and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=5 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A-D) Representative western blot of (A) lysates from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of PPIA (B) , low-glycosylated (37kDa) (C) and high-glycosylated (50kDa) (D) forms of EMMPRIN (EMN). Data are mean±SEM of n=3 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (E-G) Representative western blot of (E) media from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of extracellular PPIA (ePPIA) (F) and soluble EMMPRIN (sEMN) (G) . Data are mean±SEM of n=3-4 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (H) Luciferase assay for NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 72h and treated with 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3 independent experiments expressed as fold of Mock untreated cells. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: Target protein intensity was normalized on total transferred proteins (TTP). Relative luminescence units (RLU) were normalized on total proteins (TP, μg). *, p<0.05; **, p<0.01, ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative Proteomics, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in NTg and SOD1 G93A astrocytes. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01 by unpaired T-Test. (D) Relative quantification of factors released in 24h conditioned medium from SOD1 G93A astrocytes (G:U) compared to NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 3. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes compared to untreated NTg astrocytes. (F) Venn diagram showing 42 commonly secreted proteins between NTg astrocytes treated with PPIA (N:P) and SOD1 G93A astrocytes untreated (G:U). List of common proteins is present in Supplementary Table 4. (G) Significant leading pathways related to the 62 upregulated proteins found in SOD1 G93A astrocytes. Pathways found also in NTg astrocytes treated with PPIA are labelled with a star. Proteins associated with the pathways are listed in Supplementary Table 5.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in SOD1 G93A astrocytes treated 24h with 0.5nM anti-EMMPRIN (EMN Ab) or isotype control (CTR Ab) antibodies. Data are mean±SEM of n=3-4 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01, ***, p<0.001 by unpaired T-Test. (D) Relative quantification of the 42 commonly secreted factors between untreated SOD1 G93A and NTg astrocytes treated with PPIA (List in Supplementary Table 4) released from SOD1 G93A astrocytes after 24h treatment with 0.5nM anti-EMMPRIN (G:E) or isotype control (G:C) antibodies. Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=4 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05; ** versus SOD1 G93A astrocytes treated with control antibody by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 6. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes treated with anti-EMMPRIN compared to control antibody treated SOD1 G93A astrocytes. (F) Significant leading pathways related to the 30 downregulated proteins found in SOD1 G93A astrocytes after anti-EMMPRIN treatment. Proteins associated with the pathways are listed in Supplementary Table 7.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT , TDP-43 A315T , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=6-8 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human TDP-43 WT or TDP-43 A315T and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=3 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT or TDP-43 A315T plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or 0.5nM anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-6 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: Toxicology letters

Article Title: Dichotomous effect of methylphenidate on microglia and astrocytes: Insights from in vitro and animal studies.

doi: 10.1016/j.toxlet.2023.10.008

Figure Lengend Snippet: Fig. 5. Impact of MPH on astrocytic properties. MPH-induced production of (A) ROS and (B) NO by astrocytes. Cells were incubated with different concentrations of MPH (0.1, 0.5, and 1 mM), and to 250 μM H2O2 (positive control). (C, D) Upregulation of (C) iNOS and (D) TNF protein levels after incubation with MPH (0.5 and 1 mM). Western blot images of iNOS (130 kDa), TNF (18 kDa), and GAPDH (37 kDa) are shown. (E) Representative fluorescence images of MPH-induced p65 translocation to nuclei, indicating NF-κB pathway activation. White arrows indicate p65-positive nuclei (green). Nuclei were stained with Hoechst (blue). Scale bar = 100 µm. (F) MPH (0.5 mM) significantly increased the number of p65-positive nuclei. TNF (10 ng/mL) treatment was used as positive control and activated NF-κB in around 70% of astrocytes. Data are presented as the mean ± SEM, n = 12–18 (CellROX and DAF), n = 6–10 (western blotting), and n = 10–12 (p65 translocation). * P < 0.05, * * P < 0.01, * ** P < 0.001, and * ** * P < 0.0001 were significantly different from the control (CTR) using the Kruskal-Wallis test followed by Dunn’s multiple comparison test, or Mann-Whitney test.

Article Snippet: Afterward, cells were incubated with a rabbit primary antibody against nuclear factor-kB (NF-kB) p65 subunit (1:400, Cell Signaling Technology, MA, USA) overnight at 4 ◦C, washed with PBS and co-incubated for 1 h at RT with the secondary antibody anti-rabbit Alexa Fluor 488 (1:200, Invitrogen) and Hoechst 33342 (2 μg/mL, Sigma-Aldrich).

Techniques: Incubation, Positive Control, Western Blot, Fluorescence, Translocation Assay, Activation Assay, Staining, Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Suppressor of Cytokine Signaling 3 Derived Peptide as a Therapeutic for Inflammatory, and Oxidative Stress Induced Damage to the Retina

doi: 10.1101/2023.09.04.556227

Figure Lengend Snippet: A . ARPE-19 cells in serum free medium were treated with R9-SOCS3-KIR or the control peptide (20 μM) for 1 hr followed by treatment with C5a (50 ng/ml) for 4 hr. They were then co-transfected with a mixture of plasmids with NF-kB promoter driven firefly luciferase and another plasmid with thymidine kinase driven Renilla luciferase as an internal control and grown for 24 hrs in 1% FBS containing medium. Cell lysates, were harvested and used to measure relative luciferase units using a Dual luciferase kit from Promega. **, p = 0.001. Error bars indicae the standard deviation. Abbrev: K, R9-SOCS3-KIR, Ctrl: R9-SOCS3-KIR-scrambled peptide. B. R9-SOCS3-KIR suppressed C5a-induced nuclear translocation of NF-κB subunit p65. ARPE-19 cells were grown overnight in 8 well plates, placed in serum-free medium and treated with R9-SOCS3-KIR or its control peptide at 20 μM for 1 hr, followed by addition of C5a (50 ng/ml) for 30 min. Cells were stained with an antibody to p65, followed by staining with Cy-3 conjugated secondary antibody and DAPI, and imaged in a fluorescence microscope using the same exposure time and light intensity. Scale bars equal 50 μm.

Article Snippet: Rabbit polyclonal antibody for the NF-kB p65 subunit (Cell Signaling Technology) at 1:200 dilution was added and incubated overnight at 4 °C, followed by washing four times with the wash buffer.

Techniques: Transfection, Luciferase, Plasmid Preparation, Standard Deviation, Translocation Assay, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Suppressor of Cytokine Signaling 3 Derived Peptide as a Therapeutic for Inflammatory, and Oxidative Stress Induced Damage to the Retina

doi: 10.1101/2023.09.04.556227

Figure Lengend Snippet: J774A.1 cells were seeded in 8-well cell culture slide and grown overnight. Cells were transferred to serum free medium and pre-treated with R9-SOCS3-KIR peptide, or its control peptide at 20 µM for 1 hr, followed by treatment with LPS (1μg/ml) for 30 min. Cells were stained with an antibody to p65, the active subunit of NF-κB ( A ), or p-p38 ( B ). Secondary antibodies conjugated to Alexa-488 ( A , green), or Cy-3 red ( B , red) and DAPI (nuclei, bottom row in A and B) were used and imaged in a fluorescence microscope. Scale bar, 50 μm.

Article Snippet: Rabbit polyclonal antibody for the NF-kB p65 subunit (Cell Signaling Technology) at 1:200 dilution was added and incubated overnight at 4 °C, followed by washing four times with the wash buffer.

Techniques: Cell Culture, Staining, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Oscillating Glucose Induces the Increase in Inflammatory Stress through Ninjurin-1 Up-Regulation and Stimulation of Transport Proteins in Human Endothelial Cells.

doi: 10.3390/biom13040626

Figure Lengend Snippet: Figure 7. Inhibitors for oxidative stress and NF-kB reduce inflammation in ECs exposed to oscillating glucose. Cells were exposed alternatively to 5 mM/25 mM glucose (OG) at every 3 h, for 72 h. N-acetyl-cysteine (NAC, 7.5 mM) or Bay11-7085 (Bay, 15 µM) were added to the OG experimental condition, for the whole period of incubation. (a,d,e) Protein expression of Ninj-1 (a), TNFR1 (d) and RAGE (e) in EC lysates relative to β-actin (representative blot and densitometric analysis); (b) monocyte adhesion to endothelial cells; (c) secreted MCP-1 in the culture media relative to total cell protein (representative blots and densitometric analysis). All data are expressed as fold change versus OG and presented as mean ± SD. & p < 0.05, && p < 0.01, &&& p < 0.001 vs. OG.

Article Snippet: Antibodies to human glucose regulated protein 78 (GRP78, sc-58774), phospho-eukaryotic Initiation Factor 2α (p-eIF2α, sc-101670), total eIF2α (t-eiF2α, sc-11386), p65 nuclear factor-kB (NF-kB) subunit (sc-372), lamin B1 (sc-377000), phospho-p38 mitogen-activated protein kinase (MAPK) (pp38, sc-17852-R), total p38 MAPK (t-p38, sc-7149), Ninj-1 (sc-136295), TNFR1 (sc-8436), VAMP-3 (sc-514843), Cav-1 (sc-53564), heme oxygenase 1 (HO-1, sc-390991) and β-actin (sc-47778) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Incubation, Expressing

Journal: Biomolecules

Article Title: Oscillating Glucose Induces the Increase in Inflammatory Stress through Ninjurin-1 Up-Regulation and Stimulation of Transport Proteins in Human Endothelial Cells.

doi: 10.3390/biom13040626

Figure Lengend Snippet: Figure 8. Inhibitors for oxidative stress and NF-kB reduce the expression of Cav-1 and VAMP-3 in ECs exposed to oscillating glucose. Cells were exposed alternatively to 5 mM/25 mM glucose (OG) at every 3 h, for 72 h. N-acetyl-cysteine (NAC, 7.5 mM) or Bay11-7085 (Bay, 15 µM) were added to the OG experimental condition, for the whole period of incubation. (a–c) Protein expression of SR-BI (a), Cav-1 (b) and VAMP-3 (c) in EC lysates relative to β-actin (representative blot and densitometric analysis). All data are expressed as fold change versus OG and presented as mean ± SD. & p < 0.05 vs. OG.

Article Snippet: Antibodies to human glucose regulated protein 78 (GRP78, sc-58774), phospho-eukaryotic Initiation Factor 2α (p-eIF2α, sc-101670), total eIF2α (t-eiF2α, sc-11386), p65 nuclear factor-kB (NF-kB) subunit (sc-372), lamin B1 (sc-377000), phospho-p38 mitogen-activated protein kinase (MAPK) (pp38, sc-17852-R), total p38 MAPK (t-p38, sc-7149), Ninj-1 (sc-136295), TNFR1 (sc-8436), VAMP-3 (sc-514843), Cav-1 (sc-53564), heme oxygenase 1 (HO-1, sc-390991) and β-actin (sc-47778) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Expressing, Incubation

Journal: Biomolecules

Article Title: Oscillating Glucose Induces the Increase in Inflammatory Stress through Ninjurin-1 Up-Regulation and Stimulation of Transport Proteins in Human Endothelial Cells.

doi: 10.3390/biom13040626

Figure Lengend Snippet: Figure 11. Proposed mechanisms by which oscillating glucose (OG) generates EC dysfunction (ECD). OG determines advanced ECD through a complex process: (1) it enhances intracellular inflammatory stress, determining an increase of Ninj-1, MCP-1 and monocyte adhesion; (2) it stimulates the expression of RAGE and TNFR1; (3) it promotes the up-regulation of SR-BI and VAMP-3, the proteins involved in LDL transendothelial transport, an important process in atheroma formation. These processes are stimulated through mechanisms dependent on the increase of oxidative stress (through VPO1 and p22phox stimulation) and the nuclear translocation of NF-kB. Importantly, Ninj-1 seems to play an intermediate role between inflammation and transendothelial transport.

Article Snippet: Antibodies to human glucose regulated protein 78 (GRP78, sc-58774), phospho-eukaryotic Initiation Factor 2α (p-eIF2α, sc-101670), total eIF2α (t-eiF2α, sc-11386), p65 nuclear factor-kB (NF-kB) subunit (sc-372), lamin B1 (sc-377000), phospho-p38 mitogen-activated protein kinase (MAPK) (pp38, sc-17852-R), total p38 MAPK (t-p38, sc-7149), Ninj-1 (sc-136295), TNFR1 (sc-8436), VAMP-3 (sc-514843), Cav-1 (sc-53564), heme oxygenase 1 (HO-1, sc-390991) and β-actin (sc-47778) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Expressing, Translocation Assay