β-actin Search Results


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  • 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
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    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
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    97
    Santa Cruz Biotechnology β actin
    FMDV internalization and replication in BHK-21 cells are Cav-1 independent but require plasma membrane cholesterol. (A,B) Cav-1 downregulation did not affect the internalization of FMDV. Cells were transfected with control siRNA (left panels) or Cav-1 siRNA to downregulate Cav-1 expression (right panels). The effect of siRNA on AF594–CTxB uptake was apparent (red; upper panels). FMDV (MOI 25) was allowed to bind to siRNA-transfected cells for 1 h at 4 °C and then transferred to 37 °C. After incubation for 1 h at 37 °C, the fixed cells were processed for confocal microscopy (lower panels). (B) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. The internalized FMDV were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cav-1 downregulation did not affect the synthesis of viral proteins. The siRNA-transfected cells were infected (MOI 1) for 4 h and analyzed with an anti-FMDV antibody in Western blot, and <t>β-actin</t> was measured as the internal control. The relative quantification of the viral proteins was determined by densitometry as shown in the histogram. (D) MβCD inhibited the internalization of FMDV, whereas Nys did not. Cells were pretreated with MβCD (10 mM) or Nys (20 μg/mL) and then infected (MOI 25) as described in the Materials and Methods. Samples were then processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in Mock-, Nys- or MβCD-treated cells. (F) Nys did not affect FMDV entry and replication. Cells were treated with Nys 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1) equivalent amounts of protein were analyzed in immunoblots, and fold induction was determined by densitometry. (G) MβCD inhibited FMDV entry and multiplication. MβCD was present only during treatment for 30 min before the infection (Pre) or 60 min after virus addition (Post). Samples were then processed for Western blot. SD, standard deviation; *P
    β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 51048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    99
    Millipore monoclonal anti beta actin antibody
    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
    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
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    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 27509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    94
    Millipore anti β actin antibody
    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 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
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    99
    Cell Signaling Technology Inc 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 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
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    Millipore mouse 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
    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
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    99
    Abcam 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 Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc β actin antibodies
    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 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
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    94
    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: 94/100, based on 5323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mouse anti β actin
    hESC-NSC-derived MVs increased the level of cell autophagy after H 2 O 2  stimulation. Representative western blot images showing the protein levels of LC3, Beclin-1, and P62.  β -Actin was used as an internal control (a, b). Autophagosomes were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m (c). Autophagosomes (white arrow) were measured by TEM in four groups, scale bars: 2  μ m (d). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ P
    Mouse Anti β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti β actin monoclonal antibody
    hESC-NSC-derived MVs increased the level of cell autophagy after H 2 O 2  stimulation. Representative western blot images showing the protein levels of LC3, Beclin-1, and P62.  β -Actin was used as an internal control (a, b). Autophagosomes were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m (c). Autophagosomes (white arrow) were measured by TEM in four groups, scale bars: 2  μ m (d). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ 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
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    Abcam anti β actin antibody
    hESC-NSC-derived MVs increased the level of cell autophagy after H 2 O 2  stimulation. Representative western blot images showing the protein levels of LC3, Beclin-1, and P62.  β -Actin was used as an internal control (a, b). Autophagosomes were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m (c). Autophagosomes (white arrow) were measured by TEM in four groups, scale bars: 2  μ m (d). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ 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
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    hESC-NSC-derived MVs increased the level of cell autophagy after H 2 O 2  stimulation. Representative western blot images showing the protein levels of LC3, Beclin-1, and P62.  β -Actin was used as an internal control (a, b). Autophagosomes were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m (c). Autophagosomes (white arrow) were measured by TEM in four groups, scale bars: 2  μ m (d). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ P
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    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

    FMDV internalization and replication in BHK-21 cells are Cav-1 independent but require plasma membrane cholesterol. (A,B) Cav-1 downregulation did not affect the internalization of FMDV. Cells were transfected with control siRNA (left panels) or Cav-1 siRNA to downregulate Cav-1 expression (right panels). The effect of siRNA on AF594–CTxB uptake was apparent (red; upper panels). FMDV (MOI 25) was allowed to bind to siRNA-transfected cells for 1 h at 4 °C and then transferred to 37 °C. After incubation for 1 h at 37 °C, the fixed cells were processed for confocal microscopy (lower panels). (B) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. The internalized FMDV were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cav-1 downregulation did not affect the synthesis of viral proteins. The siRNA-transfected cells were infected (MOI 1) for 4 h and analyzed with an anti-FMDV antibody in Western blot, and β-actin was measured as the internal control. The relative quantification of the viral proteins was determined by densitometry as shown in the histogram. (D) MβCD inhibited the internalization of FMDV, whereas Nys did not. Cells were pretreated with MβCD (10 mM) or Nys (20 μg/mL) and then infected (MOI 25) as described in the Materials and Methods. Samples were then processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in Mock-, Nys- or MβCD-treated cells. (F) Nys did not affect FMDV entry and replication. Cells were treated with Nys 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1) equivalent amounts of protein were analyzed in immunoblots, and fold induction was determined by densitometry. (G) MβCD inhibited FMDV entry and multiplication. MβCD was present only during treatment for 30 min before the infection (Pre) or 60 min after virus addition (Post). Samples were then processed for Western blot. SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: FMDV internalization and replication in BHK-21 cells are Cav-1 independent but require plasma membrane cholesterol. (A,B) Cav-1 downregulation did not affect the internalization of FMDV. Cells were transfected with control siRNA (left panels) or Cav-1 siRNA to downregulate Cav-1 expression (right panels). The effect of siRNA on AF594–CTxB uptake was apparent (red; upper panels). FMDV (MOI 25) was allowed to bind to siRNA-transfected cells for 1 h at 4 °C and then transferred to 37 °C. After incubation for 1 h at 37 °C, the fixed cells were processed for confocal microscopy (lower panels). (B) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. The internalized FMDV were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cav-1 downregulation did not affect the synthesis of viral proteins. The siRNA-transfected cells were infected (MOI 1) for 4 h and analyzed with an anti-FMDV antibody in Western blot, and β-actin was measured as the internal control. The relative quantification of the viral proteins was determined by densitometry as shown in the histogram. (D) MβCD inhibited the internalization of FMDV, whereas Nys did not. Cells were pretreated with MβCD (10 mM) or Nys (20 μg/mL) and then infected (MOI 25) as described in the Materials and Methods. Samples were then processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in Mock-, Nys- or MβCD-treated cells. (F) Nys did not affect FMDV entry and replication. Cells were treated with Nys 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1) equivalent amounts of protein were analyzed in immunoblots, and fold induction was determined by densitometry. (G) MβCD inhibited FMDV entry and multiplication. MβCD was present only during treatment for 30 min before the infection (Pre) or 60 min after virus addition (Post). Samples were then processed for Western blot. SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Transfection, Expressing, Incubation, Confocal Microscopy, Infection, Western Blot, Standard Deviation

    FMDV entry into BHK-21 cells activates Rac1 and depends on Dynamin II. (A) Activation of Rac1 during FMDV entry. Cells were infected (MOI 10), and Rac1 activation was measured by GST-PAK1-PBD pull-down assay. Fold induction was determined by densitometry. (B,C) Rac1 Inh inhibited FMDV entry. Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rac1 Inh-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Rac1 Inh. (D–F) Pretreated cells (Rac1 Inh) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Expression of inactive form of Rac1 inhibited FMDV infection. Transfected cells with Rac1 (WT) and Rac1 (T17N) were infected (MOI 1) for 4 h at 37 °C and analyzed by Western blot. (I) Effect of Rac1 Inh on virus entry and post-entry steps. Cells were treated with Rac1 Inh 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (J) Dynasore (Dyna) inhibited FMDV entry and multiplication. Cells were treated with indicated concentrations of Dyna as in (I) and then processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: FMDV entry into BHK-21 cells activates Rac1 and depends on Dynamin II. (A) Activation of Rac1 during FMDV entry. Cells were infected (MOI 10), and Rac1 activation was measured by GST-PAK1-PBD pull-down assay. Fold induction was determined by densitometry. (B,C) Rac1 Inh inhibited FMDV entry. Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rac1 Inh-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Rac1 Inh. (D–F) Pretreated cells (Rac1 Inh) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Expression of inactive form of Rac1 inhibited FMDV infection. Transfected cells with Rac1 (WT) and Rac1 (T17N) were infected (MOI 1) for 4 h at 37 °C and analyzed by Western blot. (I) Effect of Rac1 Inh on virus entry and post-entry steps. Cells were treated with Rac1 Inh 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (J) Dynasore (Dyna) inhibited FMDV entry and multiplication. Cells were treated with indicated concentrations of Dyna as in (I) and then processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Activation Assay, Infection, Pull Down Assay, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Expressing, Transfection, Standard Deviation

    Myosin II is required for FMDV entry in BHK-21 cells. (A,B) Bleb inhibited FMDV entry. Pretreated cells (4 μM Bleb) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Bleb-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Bleb. (C–E) Pretreated cells (Bleb) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (C), Western blot (D), and TCID50 assay (E). (F) Pretreated cells (4 μM Bleb) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Bleb on virus entry and post-entry steps. Cells were treated with Bleb 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: Myosin II is required for FMDV entry in BHK-21 cells. (A,B) Bleb inhibited FMDV entry. Pretreated cells (4 μM Bleb) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Bleb-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Bleb. (C–E) Pretreated cells (Bleb) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (C), Western blot (D), and TCID50 assay (E). (F) Pretreated cells (4 μM Bleb) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Bleb on virus entry and post-entry steps. Cells were treated with Bleb 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    Pak1 is required for FMDV entry into BHK-21cells. (A,B) FMDV activated Pak1 during early post-infection. (A) Cells were infected (MOI 10), and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot analysis. The level of total Pak1 was measured as the control. Fold induction was determined by densitometry. (B) Cells were infected (MOI 25) and processed for confocal microscopy with anti-phospho-Pak1 (green), anti-FMDV (red), and DAPI (blue). (C,D) IPA-3 inhibited FMDV entry. Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (D) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or IPA-3-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( E–H ) FMDV infection was inhibited by IPA-3. (E–G) Pretreated cells (IPA-3) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (E), Western blot (F), and TCID50 assay (G). (H) Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( C ). (I) Effect of IPA-3 on virus entry and post-entry steps. Cells were treated with IPA-3 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: Pak1 is required for FMDV entry into BHK-21cells. (A,B) FMDV activated Pak1 during early post-infection. (A) Cells were infected (MOI 10), and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot analysis. The level of total Pak1 was measured as the control. Fold induction was determined by densitometry. (B) Cells were infected (MOI 25) and processed for confocal microscopy with anti-phospho-Pak1 (green), anti-FMDV (red), and DAPI (blue). (C,D) IPA-3 inhibited FMDV entry. Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (D) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or IPA-3-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( E–H ) FMDV infection was inhibited by IPA-3. (E–G) Pretreated cells (IPA-3) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (E), Western blot (F), and TCID50 assay (G). (H) Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( C ). (I) Effect of IPA-3 on virus entry and post-entry steps. Cells were treated with IPA-3 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Western Blot, Confocal Microscopy, Indirect Immunoperoxidase Assay, Reverse Transcription Polymerase Chain Reaction, TCID50 Assay, Standard Deviation

    CME is not the only pathway for FMDV internalization into BHK-21. (A–C) CPZ moderately inhibited FMDV entry and infection. (A) Cells were pretreated with CPZ (20 μM) and maintained during infection. The effect of CPZ on Alexa Fluor 594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), cells were fixed and incubated with anti-clathrin, anti-FMDV, and DAPI to stain clathrin (green), viral particles (red), and cell nuclei (blue), respectively (upper panels). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual Mock- or CPZ-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cells were treated with CPZ at 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1), equivalent amounts of protein were analyzed by Western blot with an anti-FMDV antibody, and β-actin was used as the internal control. Fold induction was determined by densitometry. (D–F) CHC downregulation moderately inhibited FMDV entry and infection. (D) Cells were transfected with control siRNA (left panels) or CHC siRNA to downregulate CHC expression (right panels). The efficiency of CHC downregulation was analyzed by immunofluorescent staining at 36 h post-transfection (green); the effect of siRNA on AF594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), siRNA-transfected cells were processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. (F) The efficiency of CHC downregulation was analyzed by immunoblotting. After 4 hpi (FMDV, MOI 1), the siRNA-transfected cells were processed for Western blot. SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: CME is not the only pathway for FMDV internalization into BHK-21. (A–C) CPZ moderately inhibited FMDV entry and infection. (A) Cells were pretreated with CPZ (20 μM) and maintained during infection. The effect of CPZ on Alexa Fluor 594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), cells were fixed and incubated with anti-clathrin, anti-FMDV, and DAPI to stain clathrin (green), viral particles (red), and cell nuclei (blue), respectively (upper panels). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual Mock- or CPZ-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cells were treated with CPZ at 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1), equivalent amounts of protein were analyzed by Western blot with an anti-FMDV antibody, and β-actin was used as the internal control. Fold induction was determined by densitometry. (D–F) CHC downregulation moderately inhibited FMDV entry and infection. (D) Cells were transfected with control siRNA (left panels) or CHC siRNA to downregulate CHC expression (right panels). The efficiency of CHC downregulation was analyzed by immunofluorescent staining at 36 h post-transfection (green); the effect of siRNA on AF594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), siRNA-transfected cells were processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. (F) The efficiency of CHC downregulation was analyzed by immunoblotting. After 4 hpi (FMDV, MOI 1), the siRNA-transfected cells were processed for Western blot. SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Incubation, Staining, Western Blot, Transfection, Expressing, Confocal Microscopy, Standard Deviation

    EIPA inhibits FMDV entry into BHK-21 cells and FMDV stimulates fluid-phase uptake. (A) EIPA inhibited FMDV entry. Pretreated cells (40 μM EIPA) were infected (MOI 25) for 1 h at 37 °C and then processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or EIPA-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C–E) Pretreated cells (EIPA) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (0.2 μM EIPA) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of EIPA on virus entry and post-entry steps. Cells were treated with EIPA 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (H) FMDV stimulated fluid-phase uptake. Cells were pretreated (40 μM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with AF594-dextran for 15 min, and analyzed by FACS. (I) FMDV colocalized with dextran. FMDV (MOI 25) was allowed to bind to cells for 1 h at 4 °C. The inoculum was replaced with medium containing AF594-dextran and incubated for 15 min in 37 °C. Cells were fixed and incubated with anti-FMDV antibody (green). 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: EIPA inhibits FMDV entry into BHK-21 cells and FMDV stimulates fluid-phase uptake. (A) EIPA inhibited FMDV entry. Pretreated cells (40 μM EIPA) were infected (MOI 25) for 1 h at 37 °C and then processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or EIPA-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C–E) Pretreated cells (EIPA) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (0.2 μM EIPA) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of EIPA on virus entry and post-entry steps. Cells were treated with EIPA 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (H) FMDV stimulated fluid-phase uptake. Cells were pretreated (40 μM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with AF594-dextran for 15 min, and analyzed by FACS. (I) FMDV colocalized with dextran. FMDV (MOI 25) was allowed to bind to cells for 1 h at 4 °C. The inoculum was replaced with medium containing AF594-dextran and incubated for 15 min in 37 °C. Cells were fixed and incubated with anti-FMDV antibody (green). 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, FACS, Incubation, Standard Deviation

    FMDV entry into BHK-21 cells depends on actin dynamics and induces actin ruffles. (A) FMDV entry induced actin ruffles. Cells were incubated with FMDV (MOI 100) for 1 h at 37 °C prior to incubation for 5 min at 37 °C, fixed, and processed for TEM. The arrow indicates a virion. The arrowhead indicates the membrane ruffle. (B,C) Jas inhibited FMDV entry. Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Jas-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Jas. (D–F) Pretreated cells (Jas) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Effect of Jas on virus entry and post-entry steps. Cells were treated with Jas 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: FMDV entry into BHK-21 cells depends on actin dynamics and induces actin ruffles. (A) FMDV entry induced actin ruffles. Cells were incubated with FMDV (MOI 100) for 1 h at 37 °C prior to incubation for 5 min at 37 °C, fixed, and processed for TEM. The arrow indicates a virion. The arrowhead indicates the membrane ruffle. (B,C) Jas inhibited FMDV entry. Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Jas-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Jas. (D–F) Pretreated cells (Jas) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Effect of Jas on virus entry and post-entry steps. Cells were treated with Jas 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Incubation, Transmission Electron Microscopy, Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    RTKs are required for FMDV entry into BHK-21 cells. (A,B) Gen inhibited FMDV entry. Pretreated cells (60 μM Gen) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Gen-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Gen. (C–E) Pretreated cells (Gen) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (60 μM Gen) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Gen on virus entry and post-entry steps. Cells were treated with Gen 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: RTKs are required for FMDV entry into BHK-21 cells. (A,B) Gen inhibited FMDV entry. Pretreated cells (60 μM Gen) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Gen-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Gen. (C–E) Pretreated cells (Gen) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (60 μM Gen) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Gen on virus entry and post-entry steps. Cells were treated with Gen 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    PKC is required for FMDV entry and multiplication in BHK-21 cells. (A,B) Rott inhibited FMDV entry. Pretreated cells (20 μM Rott) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rott-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Rott. (C–E) Pretreated cells (Rott) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (20 μM Rott) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Rott on virus entry and post-entry steps. Cells were treated with Rott 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: PKC is required for FMDV entry and multiplication in BHK-21 cells. (A,B) Rott inhibited FMDV entry. Pretreated cells (20 μM Rott) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rott-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Rott. (C–E) Pretreated cells (Rott) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (20 μM Rott) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Rott on virus entry and post-entry steps. Cells were treated with Rott 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    PI3K is not required for FMDV entry and replication in BHK-21 cells. (A,B) Wort moderately stimulated FMDV entry. Pretreated cells (40 μM Wort) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Wort-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–E) Wort enhanced FMDV infection. Pretreated cells (Wort) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Effect of Wort on virus entry and post-entry steps. Cells were treated with Wort 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (G) PI3K downregulation moderately enhanced FMDV infection. (H) PI3K overexpression did not affect FMDV infection. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: PI3K is not required for FMDV entry and replication in BHK-21 cells. (A,B) Wort moderately stimulated FMDV entry. Pretreated cells (40 μM Wort) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Wort-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–E) Wort enhanced FMDV infection. Pretreated cells (Wort) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Effect of Wort on virus entry and post-entry steps. Cells were treated with Wort 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (G) PI3K downregulation moderately enhanced FMDV infection. (H) PI3K overexpression did not affect FMDV infection. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Over Expression, Standard Deviation

    Eliminating expression of GLUT1 in G1fP cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP, CK18, and β-actin in lysates from G LUT 1 fl/ f l mammary cells transformed with P yVMT (G1fP cells) 72 hours after being infected with adenovirus expressing GFP (Ad-GFP) or Cre recombinase (Ad-Cre) at an MOI of 100. B–D. Uptake of 3 H-2-deoxyglucose (B) , glucose consumption ( C ), and lactate secretion ( D ) by G1fP cells previously infected with Ad-GFP or Ad-Cre as described in figure 2 . E. Proliferation is estimated by determining the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F. The concentration of ATP in the two groups of cells was determined and normalized to the DNA content of parallel monolayers. G. Lipid synthesis in G1fP cells was measured as described in figure 2 . H. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 normalized to RPL32 expression in G1fP cells that had been infected two weeks prior with Ad-GFP or Ad-Cre.

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Eliminating expression of GLUT1 in G1fP cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP, CK18, and β-actin in lysates from G LUT 1 fl/ f l mammary cells transformed with P yVMT (G1fP cells) 72 hours after being infected with adenovirus expressing GFP (Ad-GFP) or Cre recombinase (Ad-Cre) at an MOI of 100. B–D. Uptake of 3 H-2-deoxyglucose (B) , glucose consumption ( C ), and lactate secretion ( D ) by G1fP cells previously infected with Ad-GFP or Ad-Cre as described in figure 2 . E. Proliferation is estimated by determining the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F. The concentration of ATP in the two groups of cells was determined and normalized to the DNA content of parallel monolayers. G. Lipid synthesis in G1fP cells was measured as described in figure 2 . H. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 normalized to RPL32 expression in G1fP cells that had been infected two weeks prior with Ad-GFP or Ad-Cre.

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, In Vitro, Transformation Assay, Infection, Concentration Assay, Real-time Polymerase Chain Reaction

    Overexpression of GLUT1 in 85815GL cells accelerates tumor formation. A. 0.4 million 85815GL cells expressing control vector (V) or GLUT1 (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 3, 6, 9, 12 and 14 after implantation. B. The bioluminescence on days 3, 6, 9, 12 and 14 normalized to the day 3 value was averaged for all five mice and is presented +/−SEM. C. GLUT1 and β-actin expression evaluated in lysates of three tumor pairs by immunoblot analysis. D. GLUT1 expression evaluated by IHC in two tumor pairs. E. Low power photomicrographs of a pair of tumor sections derived from a vector control tumor (top) and a tumor overexpressing GLUT1 (bottom) immunostained for cleaved caspase 3 (left). High power photomicrographs of a pair of tumor sections immunostained for cleaved caspase 3 (middle), which are converted to binary pictures (right). F. Quantification of the number of cleaved caspase 3 positive pixels in five high power field “hot spots” in four tumors of each group. G–H. Representative high power photomicrographs of tumor sections immunostained for BrdU with hematoxylin counterstain ( G ) which is quantified ( H ).

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Overexpression of GLUT1 in 85815GL cells accelerates tumor formation. A. 0.4 million 85815GL cells expressing control vector (V) or GLUT1 (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 3, 6, 9, 12 and 14 after implantation. B. The bioluminescence on days 3, 6, 9, 12 and 14 normalized to the day 3 value was averaged for all five mice and is presented +/−SEM. C. GLUT1 and β-actin expression evaluated in lysates of three tumor pairs by immunoblot analysis. D. GLUT1 expression evaluated by IHC in two tumor pairs. E. Low power photomicrographs of a pair of tumor sections derived from a vector control tumor (top) and a tumor overexpressing GLUT1 (bottom) immunostained for cleaved caspase 3 (left). High power photomicrographs of a pair of tumor sections immunostained for cleaved caspase 3 (middle), which are converted to binary pictures (right). F. Quantification of the number of cleaved caspase 3 positive pixels in five high power field “hot spots” in four tumors of each group. G–H. Representative high power photomicrographs of tumor sections immunostained for BrdU with hematoxylin counterstain ( G ) which is quantified ( H ).

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Injection, Mouse Assay, Labeling, Immunohistochemistry, Derivative Assay

    Elimination of GLUT1 expression in G1fPt cells decreases tumor growth. A. 0.5 million G1fPt cells infected two weeks prior with adenovirus expressing GFP or Cre recombinase were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 12, 15 and 20 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 12, 15 and 20 +/− SEM for eight mice is presented with green diamonds representing “GFP” tumors and light blue squares representing “Cre” tumors. C. Expression of GLUT1 and β-actin in lysates of two tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for Ki67 with hematoxylin counterstain ( F ) which is quantified ( G ).

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Elimination of GLUT1 expression in G1fPt cells decreases tumor growth. A. 0.5 million G1fPt cells infected two weeks prior with adenovirus expressing GFP or Cre recombinase were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 12, 15 and 20 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 12, 15 and 20 +/− SEM for eight mice is presented with green diamonds representing “GFP” tumors and light blue squares representing “Cre” tumors. C. Expression of GLUT1 and β-actin in lysates of two tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for Ki67 with hematoxylin counterstain ( F ) which is quantified ( G ).

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, Infection, Injection, Mouse Assay, Labeling, Luciferase, Activity Assay, Immunohistochemistry

    GLUT1 is the most abundantly expressed GLUT family member in MMTV-c-ErbB2 tumors and a number of mouse mammary carcinoma cell lines. A. qPCR analysis to determine the expression of GLUT1–GLUT6, GLUT8–GLUT10, GLUT12–GLUT13 (eleven of the twelve mouse GLUT family transporters) relative to β-actin expression was performed with the cDNA equivalent of 50 ng RNA in six samples: mammary tumor from a MMTV-c-ErbB2 mouse (ErbB2 tumor), two different cell lines derived from these tumors (78617 and 85815), a cell line derived from a MMTV-PyVMT mouse mammary tumor (Met1), a cell line derived from a BALB/c mouse mammary tumor (4T1) and immortalized mouse mammary epithelial cells (EPH4). B. Quantitation of the number of copies of GLUT1, GLUT6, GLUT8 and GLUT9 RNA in the cDNA derived from 50 ng RNA from triplicate samples of ErbB2 Tumors, 78617 cells and 85815 cells. * indicates p

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: GLUT1 is the most abundantly expressed GLUT family member in MMTV-c-ErbB2 tumors and a number of mouse mammary carcinoma cell lines. A. qPCR analysis to determine the expression of GLUT1–GLUT6, GLUT8–GLUT10, GLUT12–GLUT13 (eleven of the twelve mouse GLUT family transporters) relative to β-actin expression was performed with the cDNA equivalent of 50 ng RNA in six samples: mammary tumor from a MMTV-c-ErbB2 mouse (ErbB2 tumor), two different cell lines derived from these tumors (78617 and 85815), a cell line derived from a MMTV-PyVMT mouse mammary tumor (Met1), a cell line derived from a BALB/c mouse mammary tumor (4T1) and immortalized mouse mammary epithelial cells (EPH4). B. Quantitation of the number of copies of GLUT1, GLUT6, GLUT8 and GLUT9 RNA in the cDNA derived from 50 ng RNA from triplicate samples of ErbB2 Tumors, 78617 cells and 85815 cells. * indicates p

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Quantitation Assay

    Overexpression of GLUT1 in 85815GL cells increases glucose transport without increasing proliferation. A. Expression of GLUT1, GFP-luciferase and β-actin in lysates of 85815GL cells expressing empty vector (V) or overexpressing GLUT1 (G1). B. Uptake of 3 H-2-deoxyglucose by cells expressing empty vector (Vec) or GLUT1 (GLUT1) in 15 minutes presented as CPM per µg DNA. C. Proliferation is estimated by determining the DNA content of cultures expressing empty vector or GLUT1 at days 0, 1, 2 and 3.

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Overexpression of GLUT1 in 85815GL cells increases glucose transport without increasing proliferation. A. Expression of GLUT1, GFP-luciferase and β-actin in lysates of 85815GL cells expressing empty vector (V) or overexpressing GLUT1 (G1). B. Uptake of 3 H-2-deoxyglucose by cells expressing empty vector (Vec) or GLUT1 (GLUT1) in 15 minutes presented as CPM per µg DNA. C. Proliferation is estimated by determining the DNA content of cultures expressing empty vector or GLUT1 at days 0, 1, 2 and 3.

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Over Expression, Expressing, Luciferase, Plasmid Preparation

    Reduced expression of GLUT1 in 78617GL cells decreases tumor growth. A. 0.5 million 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 2, 4, 6 and 8 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 2, 4, 6 and 8 normalized to the day 2 bioluminescence +/− SEM for five mice is presented with the black diamonds representing shCTRL tumors and grey squares representing shGLUT1 tumors. C. Expression of GLUT1 and β-actin in lysates of three tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for BrdU with hematoxylin counterstain ( F ) which is quantified ( G ).

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Reduced expression of GLUT1 in 78617GL cells decreases tumor growth. A. 0.5 million 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 2, 4, 6 and 8 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 2, 4, 6 and 8 normalized to the day 2 bioluminescence +/− SEM for five mice is presented with the black diamonds representing shCTRL tumors and grey squares representing shGLUT1 tumors. C. Expression of GLUT1 and β-actin in lysates of three tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for BrdU with hematoxylin counterstain ( F ) which is quantified ( G ).

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, shRNA, Injection, Mouse Assay, Labeling, Luciferase, Activity Assay, Immunohistochemistry

    Reduced expression of GLUT1 in 78617GL cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP-Luciferase transgene (GFP) and β-actin in lysates from 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1). B. Uptake of 3 H-2-deoxyglucose by 78617GL cells expressing control shRNA (shCTRL) or GLUT1 shRNA (shGLUT1) in 15 minutes presented as CPM per µg DNA. C–D. Glucose consumption ( C ) and lactate secretion ( D ). Glucose and lactate concentrations are normalized to the DNA content of the cultures. E. Proliferation is estimated by deteriming the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F–G. 78617GL cells were grown in soft agar for 3 weeks and colonies are pictured in F and the number of colonies per well is quantified in G . H. Quantification of BrdU positive cells in the outer edge of 12 colonies of each group. I. The concentration of ATP in the two groups of cells (lacking luciferase expression) was determined and normalized to the DNA content of parallel monolayers. J. Lipid synthesis was measured by determining the amount of 14 C in the non-aqueous chloroform fraction of methanol chloroform extracted cell lysates after 24 hour incubation with 14 C-glucose and is normalized to the DNA content of parallel samples. K. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 in 78617GL cells expressing control shRNA or GLUT1 shRNA normalized to RPL32 expression.

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Reduced expression of GLUT1 in 78617GL cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP-Luciferase transgene (GFP) and β-actin in lysates from 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1). B. Uptake of 3 H-2-deoxyglucose by 78617GL cells expressing control shRNA (shCTRL) or GLUT1 shRNA (shGLUT1) in 15 minutes presented as CPM per µg DNA. C–D. Glucose consumption ( C ) and lactate secretion ( D ). Glucose and lactate concentrations are normalized to the DNA content of the cultures. E. Proliferation is estimated by deteriming the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F–G. 78617GL cells were grown in soft agar for 3 weeks and colonies are pictured in F and the number of colonies per well is quantified in G . H. Quantification of BrdU positive cells in the outer edge of 12 colonies of each group. I. The concentration of ATP in the two groups of cells (lacking luciferase expression) was determined and normalized to the DNA content of parallel monolayers. J. Lipid synthesis was measured by determining the amount of 14 C in the non-aqueous chloroform fraction of methanol chloroform extracted cell lysates after 24 hour incubation with 14 C-glucose and is normalized to the DNA content of parallel samples. K. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 in 78617GL cells expressing control shRNA or GLUT1 shRNA normalized to RPL32 expression.

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, In Vitro, Luciferase, shRNA, Concentration Assay, Incubation, Real-time Polymerase Chain Reaction

    OTA treatment enhances autophagic flux. ( a ) PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M). At the indicated times after inoculation, the cells were collected, and the expression of p62 and  β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: OTA treatment enhances autophagic flux. ( a ) PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M). At the indicated times after inoculation, the cells were collected, and the expression of p62 and β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Inhibition of autophagy with 3-MA reverses PCV2 replication induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without 3-MA (5 mM). The cells were then assayed for ( a , b ) expression levels of LC3, Cap and  β -actin (loading control), ( c ) PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells, as described in Materials and methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy with 3-MA reverses PCV2 replication induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without 3-MA (5 mM). The cells were then assayed for ( a , b ) expression levels of LC3, Cap and β -actin (loading control), ( c ) PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells, as described in Materials and methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    Effects of OTA and/or NAC on oxidative stress and autophagy in PCV2-infected PK-15 cells. PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. ( a ) The cells were then incubated with DCFH-DA (10  μ M) at 37 °C for 30 min. The level of ROS was determined by flow cytometry. The level of intracellular ROS, which was indicated by an increase in the fluorescence intensity of the cells, was calculated as the percentage of that of the control cells. ( b ) Representative flourescent staining showing ROS visualized by DCFH-DA fluoreseence (green), and mitochondria labeled by MitoTracker Red CMXRos (red) in PK-15 cells, yellow color (green plus red) indicates the colocalization of ROS and mitochondria. Scale bar: 10  μ m. ( c ) The cells were collected, and the expression levels of LC3, ATG5, Beclin-1 and  β -actin (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. ( d ) PK-15 cells were first transfected with the GFP-LC3 plasmid. After 24 h, the cells were inoculated with PCV2 for 24 h and then incubated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10  μ m. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Effects of OTA and/or NAC on oxidative stress and autophagy in PCV2-infected PK-15 cells. PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. ( a ) The cells were then incubated with DCFH-DA (10  μ M) at 37 °C for 30 min. The level of ROS was determined by flow cytometry. The level of intracellular ROS, which was indicated by an increase in the fluorescence intensity of the cells, was calculated as the percentage of that of the control cells. ( b ) Representative flourescent staining showing ROS visualized by DCFH-DA fluoreseence (green), and mitochondria labeled by MitoTracker Red CMXRos (red) in PK-15 cells, yellow color (green plus red) indicates the colocalization of ROS and mitochondria. Scale bar: 10  μ m. ( c ) The cells were collected, and the expression levels of LC3, ATG5, Beclin-1 and β -actin (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. ( d ) PK-15 cells were first transfected with the GFP-LC3 plasmid. After 24 h, the cells were inoculated with PCV2 for 24 h and then incubated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10  μ m. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Infection, Incubation, Flow Cytometry, Cytometry, Fluorescence, Staining, Labeling, Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy

    Inhibition of autophagy with siATG5 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without ATG5 siRNA. The cells were then assayed for the expression levels of ( a ) ATG5 and  β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy with siATG5 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without ATG5 siRNA. The cells were then assayed for the expression levels of ( a ) ATG5 and β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    Inhibition of autophagy with siBeclin-1 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without Beclin-1 siRNA. Cell were assayed for expression levels of ( a ) Beclin-1 and  β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy with siBeclin-1 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without Beclin-1 siRNA. Cell were assayed for expression levels of ( a ) Beclin-1 and β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    OTA induces autophagy in PK-15 cells. ( a ) PK-15 cells were inoculated with PCV2 for 24 h, OTA was then added at concentrations of 0.01, 0.1, 1, or 2 μ M, and incubation was continued for an additional 48 h. After collecting the cells, the expression of LC3, ATG5, Beclin-1 and  β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: OTA induces autophagy in PK-15 cells. ( a ) PK-15 cells were inoculated with PCV2 for 24 h, OTA was then added at concentrations of 0.01, 0.1, 1, or 2 μ M, and incubation was continued for an additional 48 h. After collecting the cells, the expression of LC3, ATG5, Beclin-1 and β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Incubation, Expressing

    Inhibition of autophagy by CQ reverses PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without CQ (5  μ M). Cells were assayed for ( a , b ) expression levels of LC3, Cap and  β -actin (loading control), ( c )PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy by CQ reverses PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without CQ (5  μ M). Cells were assayed for ( a , b ) expression levels of LC3, Cap and β -actin (loading control), ( c )PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    Gastrodin (GAS) reduced p-tau and Aβ accumulation in the brain of Pb-treated mice. ( A ) Relative density analysis of the Aβ protein in the brain; ( B ) relative density analysis of the p-tau protein bands. The vehicle control is set as 1.0. Total tau or β-actin were probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) reduced p-tau and Aβ accumulation in the brain of Pb-treated mice. ( A ) Relative density analysis of the Aβ protein in the brain; ( B ) relative density analysis of the p-tau protein bands. The vehicle control is set as 1.0. Total tau or β-actin were probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay

    Gastrodin (GAS) increased activated Nrf2 pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Nrf2 pathway in the brain; ( B ) relative density analysis of the Nrf2 protein bands; ( C ) relative density analysis of the HO-1 protein bands; ( D ) relative density analysis of the NQO1 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) increased activated Nrf2 pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Nrf2 pathway in the brain; ( B ) relative density analysis of the Nrf2 protein bands; ( C ) relative density analysis of the HO-1 protein bands; ( D ) relative density analysis of the NQO1 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gastrodin (GAS) activated the Wnt pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Wnt pathway in the brain; ( B ) relative density analysis of the Wnt7a protein bands; ( C ) relative density analysis of the Dkk-1 protein bands; ( D ) relative density analysis of the β-catenin protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) activated the Wnt pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Wnt pathway in the brain; ( B ) relative density analysis of the Wnt7a protein bands; ( C ) relative density analysis of the Dkk-1 protein bands; ( D ) relative density analysis of the β-catenin protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gastrodin (GAS) inhibited Pb-induced apoptosis in the brain of mice. ( A ) Western blot analysis of the apoptosis-provoking proteins in the brain; ( B ) relative density analysis of the Bcl-2 protein bands; ( C ) relative density analysis of the cytochrome c in cytosol protein bands; ( D ) relative density analysis of the cleaved caspase-3 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) inhibited Pb-induced apoptosis in the brain of mice. ( A ) Western blot analysis of the apoptosis-provoking proteins in the brain; ( B ) relative density analysis of the Bcl-2 protein bands; ( C ) relative density analysis of the cytochrome c in cytosol protein bands; ( D ) relative density analysis of the cleaved caspase-3 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gastrodin (GAS) alleviated Pb-induced synaptic dysfunction in the brain of mice. ( A ) Relative density analysis of the BDNF protein bands; ( B ) relative density analysis of the NR2A protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) alleviated Pb-induced synaptic dysfunction in the brain of mice. ( A ) Relative density analysis of the BDNF protein bands; ( B ) relative density analysis of the NR2A protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay

    Gastrodin (GAS) inhibited Pb-induced inflammation in the brain of mice. ( A ) Western blot analysis of the apoptosis-related proteins in the brain; ( B ) relative density analysis of the NF-κB protein bands; ( C ) relative density analysis of the TNF-α protein bands; ( D ) relative density analysis of the COX-2 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) inhibited Pb-induced inflammation in the brain of mice. ( A ) Western blot analysis of the apoptosis-related proteins in the brain; ( B ) relative density analysis of the NF-κB protein bands; ( C ) relative density analysis of the TNF-α protein bands; ( D ) relative density analysis of the COX-2 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    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

    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

    hESC-NSC-derived MVs increased the level of cell autophagy after H 2 O 2  stimulation. Representative western blot images showing the protein levels of LC3, Beclin-1, and P62.  β -Actin was used as an internal control (a, b). Autophagosomes were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m (c). Autophagosomes (white arrow) were measured by TEM in four groups, scale bars: 2  μ m (d). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ P

    Journal: Stem Cells International

    Article Title: Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

    doi: 10.1155/2019/6452684

    Figure Lengend Snippet: hESC-NSC-derived MVs increased the level of cell autophagy after H 2 O 2 stimulation. Representative western blot images showing the protein levels of LC3, Beclin-1, and P62. β -Actin was used as an internal control (a, b). Autophagosomes were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m (c). Autophagosomes (white arrow) were measured by TEM in four groups, scale bars: 2  μ m (d). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ P

    Article Snippet: Primary antibodies, including mouse anti-β -actin (Santa Cruz Biotechnology, USA) and anti-HSP-70 (Santa Cruz Biotechnology, USA) and rabbit anti-Beclin-1, anti-P62, anti-LC3, anti-Bax, anti-Bcl-2, anti-caspase3, anti-CD63, anti-mTOR, anti-pmTOR, anti-AKT, and anti-pAkt, were purchased from Cell Signaling Technology (CST, USA).

    Techniques: Derivative Assay, Western Blot, Transmission Electron Microscopy

    Transfer of HSP-70 between cells via hESC-NSCs-derived MVs. Coomassie blue staining and western blot analysis of protein extracts from MVs (a). Western blot images showing the protein levels of the HSP-70 after HSPre with/without triptolide pretreatment (b–e). Western blot images showing the protein levels of the HSP-70 in HL-1 cells were exposed to 42°C for 10, 30, 60, and 120 min and then allowed to recover at 37°C for 0, 4, 8, 12, and 24 h.  β -Actin was used as an internal control (f–h). Every experiment was repeated at least three times. Error bars indicate mean ± SD ( ∗ P

    Journal: Stem Cells International

    Article Title: Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

    doi: 10.1155/2019/6452684

    Figure Lengend Snippet: Transfer of HSP-70 between cells via hESC-NSCs-derived MVs. Coomassie blue staining and western blot analysis of protein extracts from MVs (a). Western blot images showing the protein levels of the HSP-70 after HSPre with/without triptolide pretreatment (b–e). Western blot images showing the protein levels of the HSP-70 in HL-1 cells were exposed to 42°C for 10, 30, 60, and 120 min and then allowed to recover at 37°C for 0, 4, 8, 12, and 24 h. β -Actin was used as an internal control (f–h). Every experiment was repeated at least three times. Error bars indicate mean ± SD ( ∗ P

    Article Snippet: Primary antibodies, including mouse anti-β -actin (Santa Cruz Biotechnology, USA) and anti-HSP-70 (Santa Cruz Biotechnology, USA) and rabbit anti-Beclin-1, anti-P62, anti-LC3, anti-Bax, anti-Bcl-2, anti-caspase3, anti-CD63, anti-mTOR, anti-pmTOR, anti-AKT, and anti-pAkt, were purchased from Cell Signaling Technology (CST, USA).

    Techniques: Derivative Assay, Staining, Western Blot

    HSP-70 increased the level of cell autophagy after H 2 O 2  stimulation. (a–d) Representative western blot images showing the protein levels of LC3, Beclin-1, and P62.  β -Actin was used as an internal control. (e) Autophagosomes (white arrow) were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m. (f) Autophagosomes were measured by TEM in four groups. Every experiment was repeated at least three times, scale bars: 2  μ m. Error bars indicate mean ± SD ( ∗ P

    Journal: Stem Cells International

    Article Title: Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

    doi: 10.1155/2019/6452684

    Figure Lengend Snippet: HSP-70 increased the level of cell autophagy after H 2 O 2 stimulation. (a–d) Representative western blot images showing the protein levels of LC3, Beclin-1, and P62. β -Actin was used as an internal control. (e) Autophagosomes (white arrow) were detected by tandem fluorescent mRFG-GFP-LC3 assay, scale bars: 15  μ m. (f) Autophagosomes were measured by TEM in four groups. Every experiment was repeated at least three times, scale bars: 2  μ m. Error bars indicate mean ± SD ( ∗ P

    Article Snippet: Primary antibodies, including mouse anti-β -actin (Santa Cruz Biotechnology, USA) and anti-HSP-70 (Santa Cruz Biotechnology, USA) and rabbit anti-Beclin-1, anti-P62, anti-LC3, anti-Bax, anti-Bcl-2, anti-caspase3, anti-CD63, anti-mTOR, anti-pmTOR, anti-AKT, and anti-pAkt, were purchased from Cell Signaling Technology (CST, USA).

    Techniques: Western Blot, Transmission Electron Microscopy

    hESC-NSC-derived MVs regulate Akt and mTOR pathways by transporting HSP-70 after H 2 O 2  stimulation. (a–f) Representative western blot images showing the protein levels of AKT, p-AKT, mTOR, and p-mTOR.  β -Actin was used as an internal control. Every experiment was repeated at least three times. Error bars indicate mean ± SD ( ∗ P

    Journal: Stem Cells International

    Article Title: Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

    doi: 10.1155/2019/6452684

    Figure Lengend Snippet: hESC-NSC-derived MVs regulate Akt and mTOR pathways by transporting HSP-70 after H 2 O 2 stimulation. (a–f) Representative western blot images showing the protein levels of AKT, p-AKT, mTOR, and p-mTOR. β -Actin was used as an internal control. Every experiment was repeated at least three times. Error bars indicate mean ± SD ( ∗ P

    Article Snippet: Primary antibodies, including mouse anti-β -actin (Santa Cruz Biotechnology, USA) and anti-HSP-70 (Santa Cruz Biotechnology, USA) and rabbit anti-Beclin-1, anti-P62, anti-LC3, anti-Bax, anti-Bcl-2, anti-caspase3, anti-CD63, anti-mTOR, anti-pmTOR, anti-AKT, and anti-pAkt, were purchased from Cell Signaling Technology (CST, USA).

    Techniques: Derivative Assay, Western Blot

    hESC-NSC-derived MVs reduced cell apoptosis after H 2 O 2  stimulation. CCK-8 was used to measure HL-1 cell viability after exposure to 0, 500, 800, and 1000  μ M H 2 O 2  for 3 h (a). Representative western blot images showing the protein levels of Bcl-2, Bax, and cleaved caspase-3.  β -Actin was used as an internal control (b–f). Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining (g). The percentage of apoptotic cells was represent for both early and late apoptotic cells (h). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ P

    Journal: Stem Cells International

    Article Title: Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

    doi: 10.1155/2019/6452684

    Figure Lengend Snippet: hESC-NSC-derived MVs reduced cell apoptosis after H 2 O 2 stimulation. CCK-8 was used to measure HL-1 cell viability after exposure to 0, 500, 800, and 1000  μ M H 2 O 2 for 3 h (a). Representative western blot images showing the protein levels of Bcl-2, Bax, and cleaved caspase-3. β -Actin was used as an internal control (b–f). Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining (g). The percentage of apoptotic cells was represent for both early and late apoptotic cells (h). Every experiment was repeated at least three times; error bars indicate mean ± SD ( ∗ P

    Article Snippet: Primary antibodies, including mouse anti-β -actin (Santa Cruz Biotechnology, USA) and anti-HSP-70 (Santa Cruz Biotechnology, USA) and rabbit anti-Beclin-1, anti-P62, anti-LC3, anti-Bax, anti-Bcl-2, anti-caspase3, anti-CD63, anti-mTOR, anti-pmTOR, anti-AKT, and anti-pAkt, were purchased from Cell Signaling Technology (CST, USA).

    Techniques: Derivative Assay, CCK-8 Assay, Western Blot, Staining

    Antiapoptotic effects of HSP-70 in vitro. (a–f) Representative western blot images showing the protein levels of HSP-70, Bcl-2, Bax, and cleaved caspase-3.  β -Actin was used as an internal control. (g) Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining. (h) The percentage of apoptotic cells was represented for both early and late apoptotic cells. Every experiment was repeated at least three times. Error bars indicate mean ± SD ( ∗ P

    Journal: Stem Cells International

    Article Title: Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

    doi: 10.1155/2019/6452684

    Figure Lengend Snippet: Antiapoptotic effects of HSP-70 in vitro. (a–f) Representative western blot images showing the protein levels of HSP-70, Bcl-2, Bax, and cleaved caspase-3. β -Actin was used as an internal control. (g) Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining. (h) The percentage of apoptotic cells was represented for both early and late apoptotic cells. Every experiment was repeated at least three times. Error bars indicate mean ± SD ( ∗ P

    Article Snippet: Primary antibodies, including mouse anti-β -actin (Santa Cruz Biotechnology, USA) and anti-HSP-70 (Santa Cruz Biotechnology, USA) and rabbit anti-Beclin-1, anti-P62, anti-LC3, anti-Bax, anti-Bcl-2, anti-caspase3, anti-CD63, anti-mTOR, anti-pmTOR, anti-AKT, and anti-pAkt, were purchased from Cell Signaling Technology (CST, USA).

    Techniques: In Vitro, Western Blot, Staining