β-actin Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher β actin
    β Actin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Thermo Fisher
    Average 99 stars, based on 18135 article reviews
    Price from $9.99 to $1999.99
    β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti beta actin antibody
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
    Average 99 stars, based on 33074 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti beta actin antibody - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc β actin
    Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with <t>β-actin</t> protein. Data are expressed as means ± SD ( n = 6 sample per group). P
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 38293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 38293 article reviews
    Price from $9.99 to $1999.99
    β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Millipore anti β actin
    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. <t>β-Actin</t> was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27835 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin/product/Millipore
    Average 99 stars, based on 27835 article reviews
    Price from $9.99 to $1999.99
    anti β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    96
    Abcam β actin
    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. <t>β-Actin</t> was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 25356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Abcam
    Average 96 stars, based on 25356 article reviews
    Price from $9.99 to $1999.99
    β actin - by Bioz Stars, 2021-01
    96/100 stars
      Buy from Supplier

    99
    Millipore β actin
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 67284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Millipore
    Average 99 stars, based on 67284 article reviews
    Price from $9.99 to $1999.99
    β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    94
    Millipore anti β actin antibody
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 9783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin antibody/product/Millipore
    Average 94 stars, based on 9783 article reviews
    Price from $9.99 to $1999.99
    anti β actin antibody - by Bioz Stars, 2021-01
    94/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc anti β actin
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 9551 article reviews
    Price from $9.99 to $1999.99
    anti β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Millipore mouse anti β actin
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    Mouse Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin/product/Millipore
    Average 99 stars, based on 8890 article reviews
    Price from $9.99 to $1999.99
    mouse anti β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Abcam anti β actin
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin/product/Abcam
    Average 99 stars, based on 8076 article reviews
    Price from $9.99 to $1999.99
    anti β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc β actin antibodies
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    β Actin Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin antibodies/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1362 article reviews
    Price from $9.99 to $1999.99
    β actin antibodies - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    92
    Millipore antibodies against β actin
    Metrnl regulated AMPK phosphorylation and glucose uptake in mouse primary myoblast cells. (A) For Ca 2+ detection, myoblasts were pre‐incubated with Fluo‐3 AM (5 μ m ) for 30 min. After metrnl treatment, the Ca 2+ response was measured using a confocal microscope. Scale bars, 100 μm ( n = 5). (B) Mouse primary myoblast cells were stimulated with metrnl for the indicated times. Cell lysates were analyzed by western blotting using antibodies against phospho‐AMPKα1/2(Thr 183 /Thr 172 ) and phospho‐ACC (Ser 79 ), with AMPKα1/2, ACC, and <t>β‐actin</t> as the controls. (C) Dose‐dependent glucose uptake with metrnl treatment. Primary myoblasts were differentiated into myotubes, incubated with metrnl at several concentrations for 1 h, and then assayed for glucose uptake. Results are displayed as the mean ± SEM of five experiments. * P
    Antibodies Against β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 3619 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against β actin/product/Millipore
    Average 92 stars, based on 3619 article reviews
    Price from $9.99 to $1999.99
    antibodies against β actin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    92
    Bio-Rad β actin
    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . <t>β-actin</t> blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P
    β Actin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 5323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Bio-Rad
    Average 92 stars, based on 5323 article reviews
    Price from $9.99 to $1999.99
    β actin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    99
    Abcam anti β actin antibody
    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . <t>β-actin</t> blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P
    Anti β Actin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin antibody/product/Abcam
    Average 99 stars, based on 2512 article reviews
    Price from $9.99 to $1999.99
    anti β actin antibody - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Millipore anti β actin monoclonal antibody
    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . <t>β-actin</t> blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P
    Anti β Actin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin monoclonal antibody/product/Millipore
    Average 99 stars, based on 2002 article reviews
    Price from $9.99 to $1999.99
    anti β actin monoclonal antibody - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    93
    Abcam β actin antibody
    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . <t>β-actin</t> blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P
    β Actin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin antibody/product/Abcam
    Average 93 stars, based on 1742 article reviews
    Price from $9.99 to $1999.99
    β actin antibody - by Bioz Stars, 2021-01
    93/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti β actin antibody
    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . <t>β-actin</t> blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P
    Monoclonal Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1992 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti β actin antibody/product/Millipore
    Average 99 stars, based on 1992 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti β actin antibody - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Abcam mouse anti β actin
    Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to <t>β-actin</t> from the same sample. The data are expressed as the mean ± standard deviation. ## P
    Mouse Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin/product/Abcam
    Average 99 stars, based on 2160 article reviews
    Price from $9.99 to $1999.99
    mouse anti β actin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    N/A
    CRISPR Cas9 KO Plasmids consists of β Actin specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct the Cas9 protein
      Buy from Supplier

    N/A
    CRISPR Cas9 KO Plasmids consists of β Actin specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct the Cas9 protein
      Buy from Supplier

    N/A
    CRISPR Cas9 KO Plasmids consists of β Actin specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct the Cas9 protein
      Buy from Supplier

    N/A
    CRISPR Cas9 KO Plasmids consists of β Actin specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct the Cas9 protein
      Buy from Supplier

    Image Search Results


    Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with β-actin protein. Data are expressed as means ± SD ( n = 6 sample per group). P

    Journal: Frontiers in Physiology

    Article Title: Exercise and Omentin: Their Role in the Crosstalk Between Muscle and Adipose Tissues in Type 2 Diabetes Mellitus Rat Models

    doi: 10.3389/fphys.2018.01881

    Figure Lengend Snippet: Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with β-actin protein. Data are expressed as means ± SD ( n = 6 sample per group). P

    Article Snippet: Protein concentrations were normalized by using β-actin diluted 1:2,000 (Cell Signaling Technology, Beverly, MA, United States) in the visceral fat, or GAPDH diluted 1:10,000 (Abcam) in muscle. β-actin was detected with mouse peroxidase (HRP) – conjugated with a second antibody (Cell Signaling) diluted 1:3,000 in TBS-T, and GAPDH using antirabbit antibody (Cell Signaling), incubated and agitated for 1 h at room temperature.

    Techniques: Western Blot, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay

    Detection of histone citrullination in S . sanguinis -infected neutrophils. Neutrophils were infected with the WT, KO, or Wr strain at an MOI of 10. Following incubation for 1 h at 37°C, cells were suspended in Laemmli gel loading buffer. Samples were immunoblotted with a rabbit anti-histone H3 antibody and HRP-conjugated anti-rabbit IgG antibody (upper panel). As a loading control, β-actin was detected using an anti-β-actin antibody and HRP-conjugated anti-rabbit IgG antibody (lower panel).

    Journal: PLoS ONE

    Article Title: Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide

    doi: 10.1371/journal.pone.0172223

    Figure Lengend Snippet: Detection of histone citrullination in S . sanguinis -infected neutrophils. Neutrophils were infected with the WT, KO, or Wr strain at an MOI of 10. Following incubation for 1 h at 37°C, cells were suspended in Laemmli gel loading buffer. Samples were immunoblotted with a rabbit anti-histone H3 antibody and HRP-conjugated anti-rabbit IgG antibody (upper panel). As a loading control, β-actin was detected using an anti-β-actin antibody and HRP-conjugated anti-rabbit IgG antibody (lower panel).

    Article Snippet: As a loading control, β-actin was detected using an anti-β-actin antibody (rabbit, 1:2000, Cell Signaling, MA, USA).

    Techniques: Infection, Incubation

    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Immunoprecipitation, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

    Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, Isolation, Mouse Assay, Quantitative RT-PCR, Expressing

    Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    Rescue of KIF3A expression in Kif3a -/- MEFs does not attenuate RHOA activation mediated by purmorphamine. A . GLI1 induction and KIF3A expression in WT MEFs (WT) vs. Kif3a -/- MEFs infected with AdV-Kif3a at MOIs of 10 and 80. p38 was used as loading control (n = 3). B . Staining of primary cilia in WT MEFs, Kif3a -/- MEFs, or Kif3a -/- MEFs infected with either control (empty) AdV or AdV-Kif3a (MOI = 10). Cells were stained with anti-acetylated α-tubulin and anti-Alexa 488 to visualize the axoneme of each primary cilium. Quantification of the percentage of cilia-positive cells (mean ± S.E.M; n = 4) C . RHOA activation in Kif3a -/- MEFs infected with control AdV or AdV-Kif3a at MOI = 10 and stimulated with 5 μM PUR for the indicated times (β-actin was used as loading control because total RHOA was obscured by a cross-reactive protein that appears as a consequence of AdV infection). D . Densitometric quantification of RHOA-GTP/ β-actin increase in response to 5 μM PUR at the indicated times. The ratios were expressed as fold change compared to t = 0. * p

    Journal: PLoS ONE

    Article Title: Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia

    doi: 10.1371/journal.pone.0203170

    Figure Lengend Snippet: Rescue of KIF3A expression in Kif3a -/- MEFs does not attenuate RHOA activation mediated by purmorphamine. A . GLI1 induction and KIF3A expression in WT MEFs (WT) vs. Kif3a -/- MEFs infected with AdV-Kif3a at MOIs of 10 and 80. p38 was used as loading control (n = 3). B . Staining of primary cilia in WT MEFs, Kif3a -/- MEFs, or Kif3a -/- MEFs infected with either control (empty) AdV or AdV-Kif3a (MOI = 10). Cells were stained with anti-acetylated α-tubulin and anti-Alexa 488 to visualize the axoneme of each primary cilium. Quantification of the percentage of cilia-positive cells (mean ± S.E.M; n = 4) C . RHOA activation in Kif3a -/- MEFs infected with control AdV or AdV-Kif3a at MOI = 10 and stimulated with 5 μM PUR for the indicated times (β-actin was used as loading control because total RHOA was obscured by a cross-reactive protein that appears as a consequence of AdV infection). D . Densitometric quantification of RHOA-GTP/ β-actin increase in response to 5 μM PUR at the indicated times. The ratios were expressed as fold change compared to t = 0. * p

    Article Snippet: The β-actin antibody (Cat#A1978 clone AC-15) was purchased from Sigma-Aldrich and used at 1:10000–1:40000 dilution.

    Techniques: Expressing, Activation Assay, Infection, Staining

    HE-A, but not HE-S, reduces Aβ accumulation in APP/PS1 mice. APP/PS1 transgenic mice were orally administered with vehicle (Veh), HE-A or HE-S for 30 days ( n = 8 for each group), and then the level of Aβ 1−40 and Aβ 1−42 in cortical homogenates was determined by enzyme-linked immunosorbent assay (ELISA) ( A ). The level of APP and C-terminal fragment (CTF) ( B ), and insulin-degrading enzyme (IDE) and neprilysin (NEP) ( C ) in homogenates were analyzed by immunoblotting. The level of IDE and NEP in wild type (WT) mice ( n = 5) is also compared. Representative immunoblots are shown at the left panel. The ratio of CTF-α and −β to β-actin is presented as percentage of Veh group. The ratio of IDE to β-actin is presented as percentage of WT group. The results are the mean ± S.E.M. Significant differences between Veh group and the other groups are indicated by *, p

    Journal: International Journal of Molecular Sciences

    Article Title: The Cyanthin Diterpenoid and Sesterterpene Constituents of Hericium erinaceus Mycelium Ameliorate Alzheimer’s Disease-Related Pathologies in APP/PS1 Transgenic Mice

    doi: 10.3390/ijms19020598

    Figure Lengend Snippet: HE-A, but not HE-S, reduces Aβ accumulation in APP/PS1 mice. APP/PS1 transgenic mice were orally administered with vehicle (Veh), HE-A or HE-S for 30 days ( n = 8 for each group), and then the level of Aβ 1−40 and Aβ 1−42 in cortical homogenates was determined by enzyme-linked immunosorbent assay (ELISA) ( A ). The level of APP and C-terminal fragment (CTF) ( B ), and insulin-degrading enzyme (IDE) and neprilysin (NEP) ( C ) in homogenates were analyzed by immunoblotting. The level of IDE and NEP in wild type (WT) mice ( n = 5) is also compared. Representative immunoblots are shown at the left panel. The ratio of CTF-α and −β to β-actin is presented as percentage of Veh group. The ratio of IDE to β-actin is presented as percentage of WT group. The results are the mean ± S.E.M. Significant differences between Veh group and the other groups are indicated by *, p

    Article Snippet: The primary antibodies used were as follows: mouse anti-GFAP antibody (GFAP, Millipore, MAB360, 2041104), mouse anti-APP antibody (Millipore, MAB348, 25030193), rabbit anti-CTF antibody (Millipore, AB5352, 22051154), rabbit anti-neprilysin (NEP) antibody (Millipore, AB5458, LV1423485), rabbit anti-IDE antibody (Millipore, AB9210, 2769024), mouse anti-β-actin antibody (Millipore, MAB, LV1460528) and goat anti-Iba-1 antibody (Abcam, ab5076, GR268568-3).

    Techniques: Mouse Assay, Transgenic Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    HE-A and HE-S reduce glial activation in APP/PS1 mice. APP/PS1 transgenic mice were orally administered with vehicle (Veh), HE-A or HE-S for 30 days ( n = 8 each). Amyloid plaques were detected by immunohistochemical staining with AB-10 antibody (blue). Microglia and reactive astrocytes were detected by immunohistochemical staining with Iba-1 antibody (red) and glial fibrillary acidic protein (GFAP) antibody (green), respectively. The representative immunostaining images of clusters in section of parietal cortical area and temporal cortical area are shown ( A ). Scale bar: 500 µm. The number of microglial and astroglial cluster in the section of cerebral hemisphere is calculated by image analysis software and shown in ( B ). The representative immunostaining images of glial cells without associated with plaque in Cornu Amonis (CA)1, CA3, dentate gyrus (DG) were shown in ( C ). The image in WT mice ( n = 5) is also compared. Sale bar: 100 µm. The immunoreactivity of Iba-1 and GFAP in the CA1, CA3, and DG is calculated by image analysis software and shown in ( D ). The level of GFAP and Iba-1 in homogenates were analyzed by immunoblotting (E, F). The protein level in WT mice ( n = 5) is also compared. Representative immunoblots are shown at (E). The ratio of CTF-α and −β to β-actin is presented as percentage of Veh group. The ratio of IDE to β-actin is presented as percentage of WT group. The results are the mean ± S.E.M. Significant differences between Veh group and the other groups are indicated by *, p

    Journal: International Journal of Molecular Sciences

    Article Title: The Cyanthin Diterpenoid and Sesterterpene Constituents of Hericium erinaceus Mycelium Ameliorate Alzheimer’s Disease-Related Pathologies in APP/PS1 Transgenic Mice

    doi: 10.3390/ijms19020598

    Figure Lengend Snippet: HE-A and HE-S reduce glial activation in APP/PS1 mice. APP/PS1 transgenic mice were orally administered with vehicle (Veh), HE-A or HE-S for 30 days ( n = 8 each). Amyloid plaques were detected by immunohistochemical staining with AB-10 antibody (blue). Microglia and reactive astrocytes were detected by immunohistochemical staining with Iba-1 antibody (red) and glial fibrillary acidic protein (GFAP) antibody (green), respectively. The representative immunostaining images of clusters in section of parietal cortical area and temporal cortical area are shown ( A ). Scale bar: 500 µm. The number of microglial and astroglial cluster in the section of cerebral hemisphere is calculated by image analysis software and shown in ( B ). The representative immunostaining images of glial cells without associated with plaque in Cornu Amonis (CA)1, CA3, dentate gyrus (DG) were shown in ( C ). The image in WT mice ( n = 5) is also compared. Sale bar: 100 µm. The immunoreactivity of Iba-1 and GFAP in the CA1, CA3, and DG is calculated by image analysis software and shown in ( D ). The level of GFAP and Iba-1 in homogenates were analyzed by immunoblotting (E, F). The protein level in WT mice ( n = 5) is also compared. Representative immunoblots are shown at (E). The ratio of CTF-α and −β to β-actin is presented as percentage of Veh group. The ratio of IDE to β-actin is presented as percentage of WT group. The results are the mean ± S.E.M. Significant differences between Veh group and the other groups are indicated by *, p

    Article Snippet: The primary antibodies used were as follows: mouse anti-GFAP antibody (GFAP, Millipore, MAB360, 2041104), mouse anti-APP antibody (Millipore, MAB348, 25030193), rabbit anti-CTF antibody (Millipore, AB5352, 22051154), rabbit anti-neprilysin (NEP) antibody (Millipore, AB5458, LV1423485), rabbit anti-IDE antibody (Millipore, AB9210, 2769024), mouse anti-β-actin antibody (Millipore, MAB, LV1460528) and goat anti-Iba-1 antibody (Abcam, ab5076, GR268568-3).

    Techniques: Activation Assay, Mouse Assay, Transgenic Assay, Immunohistochemistry, Staining, Immunostaining, Software, Western Blot

    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. β-actin was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P

    Journal: Nature Communications

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice

    doi: 10.1038/s41467-018-03008-2

    Figure Lengend Snippet: CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. β-actin was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P

    Article Snippet: Antibodies against Flag-tag (M2, F3165), β-actin (A-5316), His-tag (H1029), glutamylated tubulin (B3), and GFP-tag (G-1544) were from Sigma-Aldrich (St. Louis, USA).

    Techniques: Infection, Cell Culture

    Metrnl regulated AMPK phosphorylation and glucose uptake in mouse primary myoblast cells. (A) For Ca 2+ detection, myoblasts were pre‐incubated with Fluo‐3 AM (5 μ m ) for 30 min. After metrnl treatment, the Ca 2+ response was measured using a confocal microscope. Scale bars, 100 μm ( n = 5). (B) Mouse primary myoblast cells were stimulated with metrnl for the indicated times. Cell lysates were analyzed by western blotting using antibodies against phospho‐AMPKα1/2(Thr 183 /Thr 172 ) and phospho‐ACC (Ser 79 ), with AMPKα1/2, ACC, and β‐actin as the controls. (C) Dose‐dependent glucose uptake with metrnl treatment. Primary myoblasts were differentiated into myotubes, incubated with metrnl at several concentrations for 1 h, and then assayed for glucose uptake. Results are displayed as the mean ± SEM of five experiments. * P

    Journal: The Febs Journal

    Article Title: The myokine meteorin‐like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2

    doi: 10.1111/febs.15301

    Figure Lengend Snippet: Metrnl regulated AMPK phosphorylation and glucose uptake in mouse primary myoblast cells. (A) For Ca 2+ detection, myoblasts were pre‐incubated with Fluo‐3 AM (5 μ m ) for 30 min. After metrnl treatment, the Ca 2+ response was measured using a confocal microscope. Scale bars, 100 μm ( n = 5). (B) Mouse primary myoblast cells were stimulated with metrnl for the indicated times. Cell lysates were analyzed by western blotting using antibodies against phospho‐AMPKα1/2(Thr 183 /Thr 172 ) and phospho‐ACC (Ser 79 ), with AMPKα1/2, ACC, and β‐actin as the controls. (C) Dose‐dependent glucose uptake with metrnl treatment. Primary myoblasts were differentiated into myotubes, incubated with metrnl at several concentrations for 1 h, and then assayed for glucose uptake. Results are displayed as the mean ± SEM of five experiments. * P

    Article Snippet: Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: Incubation, Microscopy, Western Blot

    Metrnl increased glucose uptake via the p38 MAPK pathway. (A) C2C12 myoblasts were stimulated for 60 min with several concentrations of metrnl. The cell lysates were analyzed by western blotting using anti‐phospho‐p38 MAPK antibody, with p38 MAPK as the control. (B) Time‐dependent phosphorylation of p38 MAPK after metrnl treatment. C2C12 myoblasts were incubated with metrnl (100 ng·mL −1 ) for the indicated times. Cell lysates were analyzed by western blotting using anti‐phospho‐p38 MAPK antibody, with p38 MAPK as the control. (C) C2C12 myoblasts were pre‐treated with compound C (10 μ m ), then treated with metrnl (100 ng·mL −1 ). Cell lysates were analyzed by western blotting using antibodies against phospho‐p38 MAPK and phospho‐AMPKα1/2(Thr 183 /Thr 172 ), with p38 MAPK and AMPKα1/2 as the controls. (D) C2C12 myoblasts were transiently transfected with AMPKα2 siRNA or non‐target siRNA. Cell lysates were analyzed by western blotting using anti‐phospho‐p38 MAPK antibody, with p38, AMPKα2, and β‐actin as the controls. (E) C2C12 myotubes were treated with metrnl (100 ng·mL −1 ) for 1 h in the presence of SB202190 (20 µ m ) and then assayed for glucose uptake. (F) C2C12 myotubes were transiently transfected with p38 MAPK siRNA or non‐target siRNA, incubated with metrnl (100 ng·mL −1 ) for 1 h, and then assayed for glucose uptake. Results are displayed as the mean ± SEM of five experiments. * P

    Journal: The Febs Journal

    Article Title: The myokine meteorin‐like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2

    doi: 10.1111/febs.15301

    Figure Lengend Snippet: Metrnl increased glucose uptake via the p38 MAPK pathway. (A) C2C12 myoblasts were stimulated for 60 min with several concentrations of metrnl. The cell lysates were analyzed by western blotting using anti‐phospho‐p38 MAPK antibody, with p38 MAPK as the control. (B) Time‐dependent phosphorylation of p38 MAPK after metrnl treatment. C2C12 myoblasts were incubated with metrnl (100 ng·mL −1 ) for the indicated times. Cell lysates were analyzed by western blotting using anti‐phospho‐p38 MAPK antibody, with p38 MAPK as the control. (C) C2C12 myoblasts were pre‐treated with compound C (10 μ m ), then treated with metrnl (100 ng·mL −1 ). Cell lysates were analyzed by western blotting using antibodies against phospho‐p38 MAPK and phospho‐AMPKα1/2(Thr 183 /Thr 172 ), with p38 MAPK and AMPKα1/2 as the controls. (D) C2C12 myoblasts were transiently transfected with AMPKα2 siRNA or non‐target siRNA. Cell lysates were analyzed by western blotting using anti‐phospho‐p38 MAPK antibody, with p38, AMPKα2, and β‐actin as the controls. (E) C2C12 myotubes were treated with metrnl (100 ng·mL −1 ) for 1 h in the presence of SB202190 (20 µ m ) and then assayed for glucose uptake. (F) C2C12 myotubes were transiently transfected with p38 MAPK siRNA or non‐target siRNA, incubated with metrnl (100 ng·mL −1 ) for 1 h, and then assayed for glucose uptake. Results are displayed as the mean ± SEM of five experiments. * P

    Article Snippet: Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: Western Blot, Incubation, Transfection

    The level of metrnl increased in vitro and in vivo exercise models. (A, B) C2C12 myotubes were subjected to an acute or chronic electrical pulse stimulation (EPS), and the conditioned media (serum‐free DMEM) were analyzed using a metrnl ELISA kit. (C) Total mRNA was prepared from C2C12 myotubes after EPS, and RT‐PCR was performed using metrnl‐specific primers. PCR products were separated on a 1% agarose gel and visualized under ultraviolet light, with β‐actin as the positive control. (D) C2C12 myotubes were subjected to acute EPS. Lysates were analyzed by western blotting using anti‐phospho‐AMPKα1/2 (Thr 183 /Thr 172 ) antibody, with AMPKα1/2 and β‐actin as the controls. (E) Total protein was prepared from C2C12 myotubes after chronic electric pulse stimulation, and western blot analysis was performed using metrnl, GLUT4, and phospho‐AMPKα1/2 (Thr 183 /Thr 172 ) antibodies, with β‐actin and AMPKα1/2 as the controls. (F) C2C12 myoblasts were transiently transfected with metrnl siRNA for 24 h. Then, the cells were subjected on acute EPS. Cell lysates were analyzed by western blotting using anti‐phospho‐AMPKα (Thr 183 /Thr 172 ), metrnl, AMPKα1/2 antibodies, with β‐actin as the controls. (G) BALB/C mice were divided into groups: sedentary ( n = 10) and forced treadmill running ( n = 10). Mice were sacrificed after chronic exercise, and the level of metrnl circulating in the blood was measured by ELISA. (H, I) Intraperitoneal (IP) GTT: blood glucose concentrations were measured after intraperitoneal administration of glucose (2 mg·kg −1 body weight). (J) Western blot analysis of phospho‐AMPKα1/2 (Thr 183 /Thr 172 ), AMPKα1/2, phosphos‐TBC1D1 (Ser 237 ), TBC1D1, and metrnl in thigh muscles of sedentary and exercise mice. β‐Actin is shown as a loading control. (K) Western blot analysis of metrnl in adipose tissues of sedentary and exercise mice. β‐Actin is shown as a loading control. Results are displayed as the mean ± SEM of five experiments. * P

    Journal: The Febs Journal

    Article Title: The myokine meteorin‐like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2

    doi: 10.1111/febs.15301

    Figure Lengend Snippet: The level of metrnl increased in vitro and in vivo exercise models. (A, B) C2C12 myotubes were subjected to an acute or chronic electrical pulse stimulation (EPS), and the conditioned media (serum‐free DMEM) were analyzed using a metrnl ELISA kit. (C) Total mRNA was prepared from C2C12 myotubes after EPS, and RT‐PCR was performed using metrnl‐specific primers. PCR products were separated on a 1% agarose gel and visualized under ultraviolet light, with β‐actin as the positive control. (D) C2C12 myotubes were subjected to acute EPS. Lysates were analyzed by western blotting using anti‐phospho‐AMPKα1/2 (Thr 183 /Thr 172 ) antibody, with AMPKα1/2 and β‐actin as the controls. (E) Total protein was prepared from C2C12 myotubes after chronic electric pulse stimulation, and western blot analysis was performed using metrnl, GLUT4, and phospho‐AMPKα1/2 (Thr 183 /Thr 172 ) antibodies, with β‐actin and AMPKα1/2 as the controls. (F) C2C12 myoblasts were transiently transfected with metrnl siRNA for 24 h. Then, the cells were subjected on acute EPS. Cell lysates were analyzed by western blotting using anti‐phospho‐AMPKα (Thr 183 /Thr 172 ), metrnl, AMPKα1/2 antibodies, with β‐actin as the controls. (G) BALB/C mice were divided into groups: sedentary ( n = 10) and forced treadmill running ( n = 10). Mice were sacrificed after chronic exercise, and the level of metrnl circulating in the blood was measured by ELISA. (H, I) Intraperitoneal (IP) GTT: blood glucose concentrations were measured after intraperitoneal administration of glucose (2 mg·kg −1 body weight). (J) Western blot analysis of phospho‐AMPKα1/2 (Thr 183 /Thr 172 ), AMPKα1/2, phosphos‐TBC1D1 (Ser 237 ), TBC1D1, and metrnl in thigh muscles of sedentary and exercise mice. β‐Actin is shown as a loading control. (K) Western blot analysis of metrnl in adipose tissues of sedentary and exercise mice. β‐Actin is shown as a loading control. Results are displayed as the mean ± SEM of five experiments. * P

    Article Snippet: Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Western Blot, Transfection, Mouse Assay

    Metrnl improved glucose tolerance in mouse models. (A) Recombinant GST‐metrnl and GST proteins were isolated using glutathione beads. The beads were washed three times with washing buffer, eluted, and analyzed by SDS/PAGE and subsequent Coomassie staining. (B) C2C12 cells were treated with recombinant GST‐metrnl. Cell lysates were analyzed with western blotting using anti‐phospho‐AMPKα1/2(Thr 183 /Thr 172 ) antibody, with AMPKα1/2 and β‐actin as the controls. (C, D) Blood glucose concentrations and area under the curve (AUC) results for the glucose tolerance test (GTT) in C57BL/6 mice injected with recombinant GST‐metrnl or GST proteins. (E, F) Blood glucose concentrations and AUC results for the GTT in db/M + , db/db + GST, and db/db + GST‐metrnl mice. (G) Fasting glucose levels results for the GTT in db/M + , db/db + GST, and db/db + GST‐metrnl mice. The mice fasted for 12 h, and tail vein blood was used to measure in the blood glucose levels. (H) Representative images of immunohistochemical detection of p‐AMPKα1/2 (Thr 183 /Thr 172 ) in the extensor digitorum longus (EDL) muscles of db/M + , db/db + GST, and db/db + GST‐metrnl mice (scale bar = 100 μm). (I, J) Blood glucose concentrations and AUC results for the GTT in mice fed an HFD or NCD in NCD‐GST, NCD‐GST‐metrnl, HFD‐GST, and HFD‐GST‐metrnl. (K) Fasting glucose levels in mice fed an HFD or NCD in NCD‐GST, NCD‐GST‐metrnl, HFD‐GST, and HFD‐GST‐metrnl. The mice fasted for 12 h and tail vein blood was used to measure in the blood glucose levels. (L) Body weight of high‐fat‐diet‐induced obesity C57BL/6 mice. Groups were compared using analysis of variance (ANOVA) with Duncan's multiple range test. Results are displayed as the mean ± SEM of five experiments. * P

    Journal: The Febs Journal

    Article Title: The myokine meteorin‐like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2

    doi: 10.1111/febs.15301

    Figure Lengend Snippet: Metrnl improved glucose tolerance in mouse models. (A) Recombinant GST‐metrnl and GST proteins were isolated using glutathione beads. The beads were washed three times with washing buffer, eluted, and analyzed by SDS/PAGE and subsequent Coomassie staining. (B) C2C12 cells were treated with recombinant GST‐metrnl. Cell lysates were analyzed with western blotting using anti‐phospho‐AMPKα1/2(Thr 183 /Thr 172 ) antibody, with AMPKα1/2 and β‐actin as the controls. (C, D) Blood glucose concentrations and area under the curve (AUC) results for the glucose tolerance test (GTT) in C57BL/6 mice injected with recombinant GST‐metrnl or GST proteins. (E, F) Blood glucose concentrations and AUC results for the GTT in db/M + , db/db + GST, and db/db + GST‐metrnl mice. (G) Fasting glucose levels results for the GTT in db/M + , db/db + GST, and db/db + GST‐metrnl mice. The mice fasted for 12 h, and tail vein blood was used to measure in the blood glucose levels. (H) Representative images of immunohistochemical detection of p‐AMPKα1/2 (Thr 183 /Thr 172 ) in the extensor digitorum longus (EDL) muscles of db/M + , db/db + GST, and db/db + GST‐metrnl mice (scale bar = 100 μm). (I, J) Blood glucose concentrations and AUC results for the GTT in mice fed an HFD or NCD in NCD‐GST, NCD‐GST‐metrnl, HFD‐GST, and HFD‐GST‐metrnl. (K) Fasting glucose levels in mice fed an HFD or NCD in NCD‐GST, NCD‐GST‐metrnl, HFD‐GST, and HFD‐GST‐metrnl. The mice fasted for 12 h and tail vein blood was used to measure in the blood glucose levels. (L) Body weight of high‐fat‐diet‐induced obesity C57BL/6 mice. Groups were compared using analysis of variance (ANOVA) with Duncan's multiple range test. Results are displayed as the mean ± SEM of five experiments. * P

    Article Snippet: Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: Recombinant, Isolation, SDS Page, Staining, Western Blot, Mouse Assay, Injection, Immunohistochemistry

    Metrnl stimulated GLUT4 translocation by AMPK‐induced TBC1D1 phosphorylation. (A) C2C12 myoblasts were stimulated for 1 h with different concentrations of metrnl. Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ) antibody, with TBC1D1 as the control. (B) C2C12 myoblasts were incubated with metrnl (100 ng·mL −1 ) for the indicated times. Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ) antibody, with TBC1D1 as the control. (C) C2C12 myoblasts were pre‐treated with compound C (10 μ m ) and then treated with metrnl (100 ng·mL −1 ). Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ) antibody, with TBC1D1 as the control. (D) C2C12 myoblasts were transiently transfected with AMPKα2 siRNA or non‐target siRNA. Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ), AMPKα2, TBC1D1 antibodies, with β‐actin as the controls. (E) C2C12 myoblasts treated with metrnl (100 ng·mL −1 ) or insulin (100 n m ) were lysed and then fractionated into the plasma membrane and cytosol. Plasma membrane (PM) and cytosol proteins were analyzed by western blotting using anti‐GLUT4 antibody, with insulin receptor (IR) as a plasma membrane marker. (F) Representative images (GLUT4, Hoechst, and merged) of cells treated with metrnl for 1 h. Insulin (100 n m ) was used as the positive control. Scale bars, 10 μm ( n = 5). (G) Surface expression of GLUT4myc with metrnl treatment. L6‐GLUT4myc myotubes were incubated with metrnl at several time points for 3 h, and then, cell surface expression of GLUT4myc was detected using an antibody‐coupled colorimetric absorbance assay. (H) L6‐GLUT4myc myotubes were treated with metrnl (100 ng·mL −1 ) for 1 h in the presence of compound C (10 µ m ), and then, cell surface expression of GLUT4myc was detected using an antibody‐coupled colorimetric absorbance assay. (I, J) L6‐GLUT4myc myotubes were transiently transfected with AMPKα2 or TBC1D1 siRNA for 48 h before metrnl (100 ng·mL −1 ) treatment for 1 h. The cell surface expression of GLUT4myc was detected using an antibody‐coupled colorimetric absorbance assay. Results are displayed as the mean ± SEM of five experiments. * P

    Journal: The Febs Journal

    Article Title: The myokine meteorin‐like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2

    doi: 10.1111/febs.15301

    Figure Lengend Snippet: Metrnl stimulated GLUT4 translocation by AMPK‐induced TBC1D1 phosphorylation. (A) C2C12 myoblasts were stimulated for 1 h with different concentrations of metrnl. Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ) antibody, with TBC1D1 as the control. (B) C2C12 myoblasts were incubated with metrnl (100 ng·mL −1 ) for the indicated times. Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ) antibody, with TBC1D1 as the control. (C) C2C12 myoblasts were pre‐treated with compound C (10 μ m ) and then treated with metrnl (100 ng·mL −1 ). Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ) antibody, with TBC1D1 as the control. (D) C2C12 myoblasts were transiently transfected with AMPKα2 siRNA or non‐target siRNA. Cell lysates were analyzed by western blotting using anti‐phospho‐TBC1D1 (Ser 237 ), AMPKα2, TBC1D1 antibodies, with β‐actin as the controls. (E) C2C12 myoblasts treated with metrnl (100 ng·mL −1 ) or insulin (100 n m ) were lysed and then fractionated into the plasma membrane and cytosol. Plasma membrane (PM) and cytosol proteins were analyzed by western blotting using anti‐GLUT4 antibody, with insulin receptor (IR) as a plasma membrane marker. (F) Representative images (GLUT4, Hoechst, and merged) of cells treated with metrnl for 1 h. Insulin (100 n m ) was used as the positive control. Scale bars, 10 μm ( n = 5). (G) Surface expression of GLUT4myc with metrnl treatment. L6‐GLUT4myc myotubes were incubated with metrnl at several time points for 3 h, and then, cell surface expression of GLUT4myc was detected using an antibody‐coupled colorimetric absorbance assay. (H) L6‐GLUT4myc myotubes were treated with metrnl (100 ng·mL −1 ) for 1 h in the presence of compound C (10 µ m ), and then, cell surface expression of GLUT4myc was detected using an antibody‐coupled colorimetric absorbance assay. (I, J) L6‐GLUT4myc myotubes were transiently transfected with AMPKα2 or TBC1D1 siRNA for 48 h before metrnl (100 ng·mL −1 ) treatment for 1 h. The cell surface expression of GLUT4myc was detected using an antibody‐coupled colorimetric absorbance assay. Results are displayed as the mean ± SEM of five experiments. * P

    Article Snippet: Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: Translocation Assay, Western Blot, Incubation, Transfection, Marker, Positive Control, Expressing

    Metrnl increased GLUT4 expression by stimulating HDAC5 phosphorylation. (A) Total mRNA from C2C12 myoblasts was prepared after metrnl (100 ng·mL −1 ) treatment for the indicated times, and real‐time qRT‐PCR was performed using GLUT4‐specific primers, with β‐actin mRNA as the positive control. (B) C2C12 myoblasts were treated with metrnl (100 ng·mL −1 ) for the indicated times. The cell lysates were analyzed by western blotting using anti‐GLUT4 antibody, with β‐actin as the control. (C) Time‐dependent phosphorylation of HDAC5 after metrnl treatment. C2C12 myoblasts were incubated with metrnl (100 ng·mL −1 ) for the indicated times. Cell lysates were analyzed by western blotting using anti‐phospho‐HDAC5 (Thr 498 ) antibody, with HDAC5 as the control. (D) C2C12 myoblasts were pre‐treated with compound C (10 μ m ) and then treated with metrnl (100 ng·mL −1 ). Cell lysates were analyzed by western blotting using anti‐phospho‐HDAC5 (Thr 498 ) antibody, with HDAC5 as the control. (E) C2C12 myoblasts were transiently transfected with AMPKα2 siRNA or non‐target siRNA. Cell lysates were analyzed by western blotting using antibodies against phospho‐HDAC5 (Thr 498 ), AMPKα2, and HDAC5, with β‐actin as the controls. (F) C2C12 myoblasts were treated with metrnl (100 ng·mL −1 ). Cytosolic and nuclear proteins were extracted from the cells. HDAC5 phosphorylation was evaluated by western blot analysis, with HDAC5 as the control. Western blotting was performed on nuclear and cytosolic fractions to detect nuclear (lamin B) and cytosolic (α‐tubulin) marker proteins. (G) Representative images of phospho‐HDAC5 treated with metrnl for 30 min. Scale bars, 10 μm ( n = 5). (H) C2C12 myoblasts were immunoprecipitated with anti‐14‐3‐3 antibody, followed by western blotting using anti‐phospho‐HDAC5, HDAC5, and 14‐3‐3 antibodies. (I) Representative images (phospho‐HDAC5 and 14‐3‐3 objective images) of cells treated with metrnl for 1 h. Scale bars, 10 μm ( n = 5). (J) The relative occupancy of HDAC5 and AcH3 on the GLUT4 promoter was assessed using a ChIP analysis following 60 min of metrnl (100 ng·mL −1 ) treatment. The ChIP data represent the ratio of IP values for each region relative to the input. The results shown are from three independent experiments. Other results are displayed as the mean ± SEM of five experiments. * P

    Journal: The Febs Journal

    Article Title: The myokine meteorin‐like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2

    doi: 10.1111/febs.15301

    Figure Lengend Snippet: Metrnl increased GLUT4 expression by stimulating HDAC5 phosphorylation. (A) Total mRNA from C2C12 myoblasts was prepared after metrnl (100 ng·mL −1 ) treatment for the indicated times, and real‐time qRT‐PCR was performed using GLUT4‐specific primers, with β‐actin mRNA as the positive control. (B) C2C12 myoblasts were treated with metrnl (100 ng·mL −1 ) for the indicated times. The cell lysates were analyzed by western blotting using anti‐GLUT4 antibody, with β‐actin as the control. (C) Time‐dependent phosphorylation of HDAC5 after metrnl treatment. C2C12 myoblasts were incubated with metrnl (100 ng·mL −1 ) for the indicated times. Cell lysates were analyzed by western blotting using anti‐phospho‐HDAC5 (Thr 498 ) antibody, with HDAC5 as the control. (D) C2C12 myoblasts were pre‐treated with compound C (10 μ m ) and then treated with metrnl (100 ng·mL −1 ). Cell lysates were analyzed by western blotting using anti‐phospho‐HDAC5 (Thr 498 ) antibody, with HDAC5 as the control. (E) C2C12 myoblasts were transiently transfected with AMPKα2 siRNA or non‐target siRNA. Cell lysates were analyzed by western blotting using antibodies against phospho‐HDAC5 (Thr 498 ), AMPKα2, and HDAC5, with β‐actin as the controls. (F) C2C12 myoblasts were treated with metrnl (100 ng·mL −1 ). Cytosolic and nuclear proteins were extracted from the cells. HDAC5 phosphorylation was evaluated by western blot analysis, with HDAC5 as the control. Western blotting was performed on nuclear and cytosolic fractions to detect nuclear (lamin B) and cytosolic (α‐tubulin) marker proteins. (G) Representative images of phospho‐HDAC5 treated with metrnl for 30 min. Scale bars, 10 μm ( n = 5). (H) C2C12 myoblasts were immunoprecipitated with anti‐14‐3‐3 antibody, followed by western blotting using anti‐phospho‐HDAC5, HDAC5, and 14‐3‐3 antibodies. (I) Representative images (phospho‐HDAC5 and 14‐3‐3 objective images) of cells treated with metrnl for 1 h. Scale bars, 10 μm ( n = 5). (J) The relative occupancy of HDAC5 and AcH3 on the GLUT4 promoter was assessed using a ChIP analysis following 60 min of metrnl (100 ng·mL −1 ) treatment. The ChIP data represent the ratio of IP values for each region relative to the input. The results shown are from three independent experiments. Other results are displayed as the mean ± SEM of five experiments. * P

    Article Snippet: Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Incubation, Transfection, Marker, Immunoprecipitation, Chromatin Immunoprecipitation

    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/mL) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Cell Culture, In Vitro, Western Blot

    Expression of Smad2 (A) and Phospho-Smad2 (B) in cultured rat decidual cells in vitro in response to TGF-β3 (ng/ml) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: Expression of Smad2 (A) and Phospho-Smad2 (B) in cultured rat decidual cells in vitro in response to TGF-β3 (ng/ml) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Cell Culture, In Vitro, Western Blot

    Expression of Smad2 (A) and Phospho-Smad2 (B) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/ml) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments.

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: Expression of Smad2 (A) and Phospho-Smad2 (B) in cultured rat decidual cells in vitro in response to TGF-β2 (ng/ml) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments.

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Cell Culture, In Vitro, Western Blot

    Expression of CDC-47 in cultured rat decidual cells in vitro in response to TGF-β2 (A) and TGF-β3 (B) (ng/ml) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. C) and D) effect of TGF-β2 and TGF-β3 (ng/ml) on cell survival in cultured rat decidual cells as demonstrated by Hoechst nuclear staining. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: Expression of CDC-47 in cultured rat decidual cells in vitro in response to TGF-β2 (A) and TGF-β3 (B) (ng/ml) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. C) and D) effect of TGF-β2 and TGF-β3 (ng/ml) on cell survival in cultured rat decidual cells as demonstrated by Hoechst nuclear staining. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Cell Culture, In Vitro, Western Blot, Staining

    TGF-β1 (A), TGF-β2 (B) and TGF-β3 (C) protein expression during the estrous cycle as determined by Western blot analysis . Total proteins extracts from rat uteri were collected at each days of the estrous cycle. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments (four different rats for each day of the estrous cycle). Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: TGF-β1 (A), TGF-β2 (B) and TGF-β3 (C) protein expression during the estrous cycle as determined by Western blot analysis . Total proteins extracts from rat uteri were collected at each days of the estrous cycle. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments (four different rats for each day of the estrous cycle). Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Western Blot

    TGF-β1 (A), TGF-β2 (B), TGF-β3 (C) and Cleaved Caspase-3 (D) proteins expression during the pseudopregnancy as determined by Western blot analysis . Total proteins extracts were collected from uteri at each days of the pseudopregnancy. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments (four different rats for each day of the pseudopregnancy). Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: TGF-β1 (A), TGF-β2 (B), TGF-β3 (C) and Cleaved Caspase-3 (D) proteins expression during the pseudopregnancy as determined by Western blot analysis . Total proteins extracts were collected from uteri at each days of the pseudopregnancy. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments (four different rats for each day of the pseudopregnancy). Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Western Blot

    Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β3 (ng/mL) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: Expression of Akt (A), Phospho-Akt (B) and XIAP (C) in cultured rat decidual cells in vitro in response to TGF-β3 (ng/mL) as demonstrated by Western blot analysis . β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent Western blot densitometrical analysis. Data represent the mean ± SEM of four independent experiments. Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Expressing, Cell Culture, In Vitro, Western Blot

    TGF-β1 (A), TGF-β2 (B) and TGF-β3 (C) mRNAs abundance during the estrous cycle as demonstrated by RT-PCR analysis . Total mRNA extracts from rat uteri were collected at each day of the estrous cycle. Data analyses were performed by the Quantity One software and are presented as a ratio (value/β-actin). β-actin blots shown were used as controls to correct for loading in each lane. Results represent the mean ± SEM of four independent experiments (four different rats for each day of the estrous cycle). Columns with different letters are significantly different from each other (P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    doi: 10.1186/1477-7827-7-80

    Figure Lengend Snippet: TGF-β1 (A), TGF-β2 (B) and TGF-β3 (C) mRNAs abundance during the estrous cycle as demonstrated by RT-PCR analysis . Total mRNA extracts from rat uteri were collected at each day of the estrous cycle. Data analyses were performed by the Quantity One software and are presented as a ratio (value/β-actin). β-actin blots shown were used as controls to correct for loading in each lane. Results represent the mean ± SEM of four independent experiments (four different rats for each day of the estrous cycle). Columns with different letters are significantly different from each other (P

    Article Snippet: Densitometrical analyses were performed (mRNA of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Software

    Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: Gastrodin protects MC3T3-E1 osteoblasts from dexamethasone-induced cellular dysfunction and promotes bone formation via induction of the NRF2 signaling pathway

    doi: 10.3892/ijmm.2018.3414

    Figure Lengend Snippet: Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Article Snippet: Rabbit anti-HO-1 (cat. no. ab68477; 1:1,000), rabbit anti-NQO-1 (cat. no. ab76956; 1:1,000), and mouse anti-β-actin (cat. no. ab8245; 1:1,000), Anti-OCN (cat. no. ab93876; 1:1,000) monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Standard Deviation