primary antibodies against mouse il6  (Stratech Scientific Ltd)

Journal: International Journal of Molecular Sciences

Article Title: The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

doi: 10.3390/ijms21124454

Figure Lengend Snippet: IL6 levels increase and correlate with miR-34a expression during vascular aging and human aortic smooth muscle cells (HASMCs) senescence. ( A ) IL6 expression in aortas of 2.5-month-old (young) and 21-month-old (old) mice analyzed by qRT-PCR and normalized to HPRT levels. Values represent the means ± SD. *, p <0.05; Mann Whitney test; n = 5 young, 3 old mice. ( B ) Correlation analysis between miR-34a and IL6 levels in mice aortas. r = Pearson’s coefficient; n = 5 young (red), 3 old (black) mice. ( C , D ) The miR-34a and IL6 mRNA expression in replicative young human aortic smooth muscle cells (HASMCs), isolated from different donors of indicated age (year-old; yo), was evaluated by qRT-PCR and normalized to corresponding U6 and GAPDH levels, respectively. Correlation analysis between ( C ) age (Age; years) and miR-34a or IL6 mRNA levels and ( D ) miR-34a and IL6 mRNA levels. r = Pearson’s coefficient; n = 3 donors. ( E , F ) miR-34a and IL6 mRNA expression in HASMCs isolated from donors of indicated age (year-old; yo) at young replicative (P5) and senescent (P15) passages evaluated by qRT-PCR and normalized to corresponding U6 and GAPDH levels, respectively. *, p <0.05; paired t test; n = 3 donors.

Article Snippet: Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber.

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Isolation

Journal: International Journal of Molecular Sciences

Article Title: The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

doi: 10.3390/ijms21124454

Figure Lengend Snippet: IL6 and IL8 expression and secretion increases upon miR-34a overexpression in HASMCs. HASMCs of 22- or 43-year-old donors were infected with either pMIRNA1 (CTRL) or pMIRH34a (miR-34a) lentivirus. ( A ) HASMCs were cultured in a growth medium for 48 hour. IL6 expression was quantified by qRT-PCR and normalized to corresponding HPRT levels. Values are mean ± SD; *, p < 0.05; Student’s t -test; n = 3–4. ( B ) HASMCs were cultured in growth medium for 48 and 72 hours (h). Amount of IL6 was quantified by ELISA assay in the supernatant of cells. Values are mean ± SD; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; 1-way ANOVA followed by Bonferroni’s multiple comparison test; n = 3–4. ( C ) HASMCs were cultured in growth medium for 48 and 72 h. Amount of IL8 was quantified by ELISA assay in the supernatant of cells. Values are mean ± SD; 22 yo, n = 5; 43 yo, n = 3.

Article Snippet: Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber.

Techniques: Expressing, Over Expression, Infection, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

doi: 10.3390/ijms21124454

Figure Lengend Snippet: Conditioned medium of miR-34a-overexpressing HASMCs enhances their senescence and calcification. Conditioned medium of HASMCs from 22- or 43-year-old (yo) donors was collected 72 h after infection with pMIRNA1 (CTRL) or pMIRH34a (miR-34a) lentivirus. ( A ) HASMCs were cultured for 24 h in the presence of corresponding conditioned medium. p21 and p16 expression was quantified by qRT-PCR and normalized to corresponding HPRT levels. Values are mean ± SD; *, p < 0.05; **, p < 0.01; Student’s t -test (22 yo); Mann–Whitney test (43 yo); n = 6. ( B , C ) HASMCs were cultured for 24 h in the presence of corresponding conditioned medium and processed for senescence-associated β-galactosidase (SA-β-gal) staining. ( B ) Representative images of SA-β-gal staining. Bar = 100 µm. (C) Bars show quantification of SA-β-gal-positive cells relative to (B). Values are mean ± SD; **, p < 0.01; ****, p < 0.0001; Student’s t -test; 22 yo, n = 3; 43 yo, n = 4. ( D ) Cells were cultured for 24 h in the presence of the corresponding conditioned medium and then in the osteogenic medium for 7 days. Calcification was measured by colorimetric analysis. Values are mean ± SD; *, p < 0.05; **, p < 0.01; ****, p < 0.0001; Student’s t -test; 22 yo, n = 6, 6; 43 yo, n = 6, 4. ( E ) HASMCs of 22- and 43 year-old (yo) donors were pretreated with indicated concentration of recombinant IL6 and subsequently cultured in osteogenic medium for 7 days. Calcification was measured by colorimetric analysis. Values are mean ± SD; *, p < 0.05; Student’s t -test; n = 3–4.

Article Snippet: Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber.

Techniques: Infection, Cell Culture, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Staining, Concentration Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

doi: 10.3390/ijms21124454

Figure Lengend Snippet: Senescence-associated secretory phenotype (SASP) factors significantly induced by overexpression of miR-34a in HASMCs.

Article Snippet: Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber.

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

doi: 10.3390/ijms21124454

Figure Lengend Snippet: IL6 expression in Mir34a +/+ and Mir34a −/− mice early after vitamin D treatment. Twelve-week-old Mir34a +/+ and Mir34a −/− mice were treated subcutaneously with either vitamin D (vit D) or a mock solution (Ctrl) for three consecutive days and sacrificed 3 days after the first injection (Day 3). ( A ) IL6 expression was analyzed by qRT-PCR and normalized to HPRT levels in the kidney and abdominal aorta. Values are mean ± SD. ** p < 0.01, *** p < 0.001; 1-way ANOVA with a Bonferroni post hoc test; n = 6, 5, 6–7 and 4–5. ( B ) Representative images of thoracic aorta sections stained for IL6 expression with a specific antibody. Bar = 20 μm. ( C ) Bars show quantification of the percentage of IL6 positive area to the total thoracic aortic area relative to ( B ). Values are mean ± SD; ***, p < 0.001; 1-way ANOVA followed by Bonferroni’s multiple comparison test; n = 4, 4, 4 and 3.

Article Snippet: Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber.

Techniques: Expressing, Injection, Quantitative RT-PCR, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

doi: 10.3390/ijms21124454

Figure Lengend Snippet: IL6 but not IL8 levels correlate with miR34a expression in healthy subjects. The expression levels of miR-34a were evaluated by qRT-PCR and IL6 or IL8 by ELISA in the serum of 20-90 years-old healthy subjects. Scatter plots showing correlation between age and miR-34a ( A ), age and IL6 ( B ), miR34a and IL6 ( C ), age and IL8 ( D ), miR-34a and IL8 ( E ). Variables showed a skewness distribution and were log-transformed. r = Pearson’s coefficient; n = 127–128.

Article Snippet: Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: Scientific Reports

Article Title: Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington’s disease by etanercept treatment

doi: 10.1038/s41598-019-43627-3

Figure Lengend Snippet: Plasma levels of TNFα and cytokines induced by TNFα are increased in late stage R6/2 mice. Increased levels of plasma TNFα, IL1β, IL2, IL6 and IL10 in late-stage R6/2 compared to WT mice at 14 weeks of age, as measured by MSD ( n = 16–26/group). Student’s t test ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies against mouse IL6 (Stratech Scientific, A00102-2-WBO) or TNFα (Bio Techne, AF-410-NA) and α-tubulin (αTub) (Sigma, MAB1637) were incubated overnight at 4 °C in BB-T. Blots were washed three times for 5 min in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and incubated for 1 hr at RT with the appropriate secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (Li-Cor) in BB-T. Blots were washed three times for 5 min in TBS-T, and the target protein band fluorescence signal quantified using the Odyssey Image Studio System (Li-Cor).

Techniques:

Journal: Scientific Reports

Article Title: Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington’s disease by etanercept treatment

doi: 10.1038/s41598-019-43627-3

Figure Lengend Snippet: A single IV dose of etanercept effectively reduced IL6 in 13–14 week old R6/2 plasma but significantly increased TNFα levels. Levels of plasma ( a ) IL6, ( b ) IL1β, ( c ) IL2, ( d ) IL10 and ( e ) TNFα following treatment of 13 week old R6/2 mice with a single IV dose of etanercept, as measured by MSD ( n = 5–9/time point). Cytokine levels were also assessed in WT littermates to determine non-disease associated levels. One-way ANOVA with Bonferroni correction ± SEM. * p < 0.05 vs R6/2 levels at day 0.

Article Snippet: Primary antibodies against mouse IL6 (Stratech Scientific, A00102-2-WBO) or TNFα (Bio Techne, AF-410-NA) and α-tubulin (αTub) (Sigma, MAB1637) were incubated overnight at 4 °C in BB-T. Blots were washed three times for 5 min in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and incubated for 1 hr at RT with the appropriate secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (Li-Cor) in BB-T. Blots were washed three times for 5 min in TBS-T, and the target protein band fluorescence signal quantified using the Odyssey Image Studio System (Li-Cor).

Techniques:

Journal: Scientific Reports

Article Title: Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington’s disease by etanercept treatment

doi: 10.1038/s41598-019-43627-3

Figure Lengend Snippet: Etanercept treatment reduced splenic Tnfα and Il6 gene expression in R6/2 mice. Gene expression of ( a ) Tnfα and ( b ) Il6 in splenocytes of 13 week old R6/2 mice treated with a single IV dose of etanercept as assessed by real-time qPCR ( n = 5/group). Student’s t test ± SEM. ** p ≤ 0.01 vs R6/2 day 0.

Article Snippet: Primary antibodies against mouse IL6 (Stratech Scientific, A00102-2-WBO) or TNFα (Bio Techne, AF-410-NA) and α-tubulin (αTub) (Sigma, MAB1637) were incubated overnight at 4 °C in BB-T. Blots were washed three times for 5 min in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and incubated for 1 hr at RT with the appropriate secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (Li-Cor) in BB-T. Blots were washed three times for 5 min in TBS-T, and the target protein band fluorescence signal quantified using the Odyssey Image Studio System (Li-Cor).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington’s disease by etanercept treatment

doi: 10.1038/s41598-019-43627-3

Figure Lengend Snippet: Multiple etanercept IP injections over a two to three week period are required to reduce plasma cytokine levels in R6/2 mice. Plasma IL10, IL12, IL1β, IL2, IL6, and TNFα levels following two ( a ) or three ( b ) weeks treatment of 9–10 week old (at the time of the first injection) R6/2 mice with IP etanercept, or PBS, injections every three days, as measured by MSD ( n = 5–8/time point). Cytokine levels were also assessed in WT littermates to determine non-disease associated levels. Two-way ANOVA with Bonferroni correction ± SEM. * p < 0.05, ** p ≤ 0.01 vs R6/2 PBS treated.

Article Snippet: Primary antibodies against mouse IL6 (Stratech Scientific, A00102-2-WBO) or TNFα (Bio Techne, AF-410-NA) and α-tubulin (αTub) (Sigma, MAB1637) were incubated overnight at 4 °C in BB-T. Blots were washed three times for 5 min in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and incubated for 1 hr at RT with the appropriate secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (Li-Cor) in BB-T. Blots were washed three times for 5 min in TBS-T, and the target protein band fluorescence signal quantified using the Odyssey Image Studio System (Li-Cor).

Techniques: Injection

Journal: Scientific Reports

Article Title: Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington’s disease by etanercept treatment

doi: 10.1038/s41598-019-43627-3

Figure Lengend Snippet: Etanercept treatment lowered the levels of soluble TNFα in the R6/2 striatum. Striatal tissue lysates of 14 week old mice, obtained three days after the final injection were immunoprobed for their TNFα and IL6 content by western blot analysis. ( a ) mTNFα and sTNFα levels normalised to αTub ± SEM. ( b ) A representative western blot for mTNFα, sTNFα and αTub. ( c ) IL6 normalised to αTub ± SEM ( d ) a representative western blot for IL6 and αTub. N = 11–12/genotype-treatment group). * p < 0.05 (vs WT-PBS), by two way ANOVA with Bonferroni correction. mTNFα = membrane-bound TNFα, sTNFα = soluble TNFα, αTub = α-tubulin, R = R6/2, W = WT, −P = PBS treated, −E = etanercept treated. Complete western blots are shown in Figs and .

Article Snippet: Primary antibodies against mouse IL6 (Stratech Scientific, A00102-2-WBO) or TNFα (Bio Techne, AF-410-NA) and α-tubulin (αTub) (Sigma, MAB1637) were incubated overnight at 4 °C in BB-T. Blots were washed three times for 5 min in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and incubated for 1 hr at RT with the appropriate secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (Li-Cor) in BB-T. Blots were washed three times for 5 min in TBS-T, and the target protein band fluorescence signal quantified using the Odyssey Image Studio System (Li-Cor).

Techniques: Injection, Western Blot