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

Journal: International Journal of Molecular Sciences

Article Title: CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

doi: 10.3390/ijms22010406

Figure Lengend Snippet: Activation of the AKT- IκB kinase (IKK)-inhibitors of NF-κB (IκB) pathway is associated with the CK2 down-regulation-mediated nuclear import of NF-κB. ( A ) MCF-7 and HCT116 cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days. The level of each protein was determined by immunoblot analysis using specific antibodies ( left ). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs represent the quantitation of p-AKT, IKK, and IκBα relative to β-actin and that of p-IKK and p-IκBα relative to the unphosphorylated proteins ( right ). ( B ) Cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days in the absence or presence of 1 μM triciribine (TCN). Cytoplasm and nuclei were isolated from cells, and both extracts were visualized by immunoblotting. α-Tubulin (cytoplasmic marker) and histone H3 (nuclear marker) were quantified as loading controls ( left ). Representative data from three independent experiments are shown. Graphs represent the quantitation of RelA/p65 relative to the subcellular markers ( right ). exp, exposure. Data are mean ± SEM. * p

Article Snippet: Materials Antibodies against SIRT1, CK2α, p53-p21Cip1/WAF1, RelA/p65, IκB, α-tubulin, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Quantitation Assay, Isolation, Marker

Journal: International Journal of Molecular Sciences

Article Title: CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

doi: 10.3390/ijms22010406

Figure Lengend Snippet: Antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 attenuate the induction of senescence-associated secretory phenotype (SASP) factors by lipopolysaccharide (LPS). ( A ) MCF-7 and HCT116 cells were transfected with the 4 miRs (miR-186, miR-216b, miR-337-3p, and miR-760) or the 4 miRi. ( B , C ) Cells were treated with LPS (6 μg/μL) in the presence or absence of the 4 miRi or pcDNA3.1-HA-CK2α for 2 days. The level of each protein was determined by immunoblot analysis using specific antibodies ( left ). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs represent the quantitation of p-AKT, IKK, and IκBα relative to β-actin and that of p-IKK and p-IκBα relative to the unphosphorylated proteins ( right ). Data are mean ± SEM. * p

Article Snippet: Materials Antibodies against SIRT1, CK2α, p53-p21Cip1/WAF1, RelA/p65, IκB, α-tubulin, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

doi: 10.3390/ijms22010406

Figure Lengend Snippet: CK2 down-regulation stimulates the expression of senescence-associated secretory phenotype (SASP) factors by enhancing the nuclear localization of NF-kB in human cancer cells. MCF-7 and HCT116 cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days. ( A ) The level of each mRNA was measured by RT-PCR using gene-specific primers ( left ). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs represent the quantitation of each mRNA relative to β-actin ( right ). ( B ) The level of each protein was determined by immunoblot analysis using specific antibodies ( left ). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs represent the quantitation of each protein relative to β-actin ( right ). ( C ) Cytoplasm and nuclei were isolated from cells, and both extracts were visualized by immunoblotting. α-Tubulin (cytoplasmic marker) and histone H3 (nuclear marker) were quantified as loading controls ( left ). Representative data from three independent experiments are shown. Graphs represent the quantitation of RelA/p65 relative to the subcellular markers ( right ). exp, exposure. Data are mean ± standard error of the mean (SEM). * p

Article Snippet: Materials Antibodies against SIRT1, CK2α, p53-p21Cip1/WAF1, RelA/p65, IκB, α-tubulin, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Isolation, Marker

Journal: International Journal of Molecular Sciences

Article Title: CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

doi: 10.3390/ijms22010406

Figure Lengend Snippet: SIRT1 attenuates both RelA/p65 acetylation and activation of the AKT-IKK-IκB axis mediated by CK2 down-regulation. ( A ) MCF-7 and HCT116 cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days in the absence or presence of 20 μM resveratrol or 15 mM nicotine amide. The level of each protein was determined by immunoblot analysis using specific antibodies ( top ). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs represent the quantitation of acetylated p65 (ac-p65) relative to unacetylated p-65 ( bottom ). RSV, resveratrol; NA, nicotine amide. ( B ) Cells were transfected with CK2α siRNA and/or pECE-Flag-SIRT1 for two days. The level of each protein was determined by immunoblot analysis using specific antibodies ( top ). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs represent the quantitation of p-AKT, IKK, and IκBα relative to β-actin and that of p-IKK and p-IκBα relative to unphosphorylated proteins ( bottom ). Data are mean ± SEM. * p

Article Snippet: Materials Antibodies against SIRT1, CK2α, p53-p21Cip1/WAF1, RelA/p65, IκB, α-tubulin, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Quantitation Assay

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

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

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

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

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

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

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

Journal: PLoS ONE

Article Title: Gp96 deficiency affects TLR4 functionality and impairs ERK and p38 phosphorylation

doi: 10.1371/journal.pone.0193003

Figure Lengend Snippet: LPS-TLR4 signalling is active in both WT and conditional LysMcre-gp96flox-mice. Peritoneal macrophages were isolated from both WT and conditional gp96-KO and treated with lipopolysaccharides (LPS) (100ng/ml) for 30, 60 and 120 minutes. A and B) Total protein was isolated after LPS treatment and Western blots performed for p-IκBα, IκBα, p-NF-κB and β-actin. Graphs show the densitometry quantification of the blots (n = 3). C) After LPS treatment, macrophages from both WT and conditional gp96-KO were permeabilized and the expression of p- NF-κB was analysed by flow cytometry. The histograms are representative of three independent experiments. D) RNA was isolated after LPS treatment and the expression of IL-6, IL-8 and TNF-α mRNA was measured by real-time quantitative PCR. “Vh” represents macrophage treated with vehicle. Graphs show the fold induction of each gene (n = 4). In all cases data are expressed as mean±SEM. Statistical analysis was performed by One-way ANOVA followed by a Newman-Keuls test. * = P

Article Snippet: The antibodies against β-actin and lamin A/C were from Millipore (Bedford, MA, USA) and BD Biosciences (Allschwil, Switzerland), respectively.

Techniques: Mouse Assay, Isolation, Western Blot, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Distinct roles for the RNA-binding protein Staufen1 in prostate cancer

doi: 10.1186/s12885-021-07844-2

Figure Lengend Snippet: Staufen1 knockdown inhibits Prostate Cancer cell migration. Migration assays were performed after 48 h of Staufen1 knockdown and a is a representative western blot of Staufen1 expression with β-actin as a loading control in PC3 and DU145 cells. Each representative blot is cropped to show an n = 1 for each cell line from their respective full-length blot. Note that the DU145 cells used for Fig. 4 were also used for Fig. 5 experiments and therefore a different representative image from the same blot was selected for each figure. Quantification is represented normalized to CTL (n = 3). Data are Mean ± SD, One-Sample T-Test, **P

Article Snippet: The antibodies used were anti-Staufen1 (ab73478, Abcam, Ontario, Canada), anti-phospho (Ser2448)-mTOR (#2971, Cell Signaling Technology, Danvers, MA, USA), anti-mTOR (#2983, Cell Signaling Technology, Danvers, MA, USA), anti-phospho (Try576/577)-FAK (#3281, Cell Signaling Technology, Danvers, MA, USA), anti-phospho (Tyr397)-FAK (#8556, Cell Signaling Technology, Danvers, MA, USA), anti-FAK (#13009, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-4E-BP1 (Thr37/46) (# 9459, Cell Signaling Technology, Danvers, MA, USA), anti-4E-BP1 (# 9452, Cell Signaling Technology, Danvers, MA, USA), HA.11 clone 16B12 (1:1000; BioLegend, California, USA), anti-β-actin (#47778, Santa Cruz Biotechnology, CA, USA), and anti-GAPDH (ab8245, Abcam, Ontario, Canada).

Techniques: Migration, Western Blot, Expressing

Journal: BMC Cancer

Article Title: Distinct roles for the RNA-binding protein Staufen1 in prostate cancer

doi: 10.1186/s12885-021-07844-2

Figure Lengend Snippet: Staufen1 Differentially Regulates Tumorigenesis via regulation of Signaling Pathways across Prostate Cancer Cell Lines. Western blot analysis was performed 48 h post-infection of prostate cancer cells with Control (CTL) or Staufen1-shRNA (shStau1) lentivirus ( a and d ) representative Staufen1 expression in LNCaP and PC3 cells, respectively. LNCaP cells were examined for ( b ) expression of total mTOR and phospho-mTOR (Ser2448) and ( c ) expression of total 4E-BP1 and phospho-4E-BP1 (Thr37/46). PC3 cells were analyzed for members of the FAK signaling pathway where ( e ) represents total FAK and phospho-FAK (Tyr397 and Tyr576/577). All quantifications are normalized to loading controls GAPDH or β-actin and represented as a fold change relative to the Control (CTL) with n = 4. Data are Mean ± SD, One-Sample T-Test, *P

Article Snippet: The antibodies used were anti-Staufen1 (ab73478, Abcam, Ontario, Canada), anti-phospho (Ser2448)-mTOR (#2971, Cell Signaling Technology, Danvers, MA, USA), anti-mTOR (#2983, Cell Signaling Technology, Danvers, MA, USA), anti-phospho (Try576/577)-FAK (#3281, Cell Signaling Technology, Danvers, MA, USA), anti-phospho (Tyr397)-FAK (#8556, Cell Signaling Technology, Danvers, MA, USA), anti-FAK (#13009, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-4E-BP1 (Thr37/46) (# 9459, Cell Signaling Technology, Danvers, MA, USA), anti-4E-BP1 (# 9452, Cell Signaling Technology, Danvers, MA, USA), HA.11 clone 16B12 (1:1000; BioLegend, California, USA), anti-β-actin (#47778, Santa Cruz Biotechnology, CA, USA), and anti-GAPDH (ab8245, Abcam, Ontario, Canada).

Techniques: Western Blot, Infection, shRNA, Expressing

Journal: BMC Cancer

Article Title: Distinct roles for the RNA-binding protein Staufen1 in prostate cancer

doi: 10.1186/s12885-021-07844-2

Figure Lengend Snippet: Staufen1 knockdown inhibits Prostate Cancer cell invasion and motility. Motility and Invasion assays were performed following 48 h of Control (CTL) or Staufen1-shRNA (shStau1) expression. a Western blot analysis of Staufen1 expression showing a representative blot of Staufen1 with β-actin as a loading control in PC3 and DU145 cells. Each representative blot is cropped to show an n = 1 for each cell line from their respective full-length blot. Note that the DU145 cells used for Fig. 4 were also used for Fig. 5 experiments and therefore a different representative image from the same blot was selected for each figure. Quantification of n = 4 is represented normalized to CTL. Data are Mean ± SD, One-Sample T-Test, **P

Article Snippet: The antibodies used were anti-Staufen1 (ab73478, Abcam, Ontario, Canada), anti-phospho (Ser2448)-mTOR (#2971, Cell Signaling Technology, Danvers, MA, USA), anti-mTOR (#2983, Cell Signaling Technology, Danvers, MA, USA), anti-phospho (Try576/577)-FAK (#3281, Cell Signaling Technology, Danvers, MA, USA), anti-phospho (Tyr397)-FAK (#8556, Cell Signaling Technology, Danvers, MA, USA), anti-FAK (#13009, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-4E-BP1 (Thr37/46) (# 9459, Cell Signaling Technology, Danvers, MA, USA), anti-4E-BP1 (# 9452, Cell Signaling Technology, Danvers, MA, USA), HA.11 clone 16B12 (1:1000; BioLegend, California, USA), anti-β-actin (#47778, Santa Cruz Biotechnology, CA, USA), and anti-GAPDH (ab8245, Abcam, Ontario, Canada).

Techniques: shRNA, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Camellioside A, isolated from Camellia japonica flowers, attenuates UVA-induced production of MMP-1 in HaCaT keratinocytes via suppression of MAPK activation

doi: 10.3892/etm.2020.9448

Figure Lengend Snippet: Effect of CMDA on the phosphorylation of MAPK and AP-1. (A) Effect of CMDA on the total and phosphorylated protein levels of p38, ERK and JNK MAPKs was analyzed in UVA-irradiated (10 J/cm 2 ) HaCaT keratinocytes after 24 h of incubation via western blotting. β-actin was used as an internal loading control. (B) Effect of CMDA on the total and phosphorylated protein levels of c-Fos and c-Jun was investigated in both total cell lysates and nuclear fractions of UVA-irradiated (10 J/cm 2 ) HaCaT keratinocytes after 24 h of incubation via western blotting. β-actin and lamin B1 were used as internal loading controls. CMDA, camellioside A; MAPK, mitogen-activated protein kinase; AP-1, activator protein 1; UVA, ultraviolet A.

Article Snippet: The following primary antibodies were used: MMP-1 (cat. no. sc-6837; Santa Cruz Biotechnology, Inc.), MMP-9 (cat. no. 393857; Cell Signaling Technology, Inc.), type I pro-collagen (cat. no. sc-8782; Santa Cruz Biotechnology, Inc.), p38 (cat. no. 8690; Cell Signaling Technology, Inc.), phosphorylated (p)-p38 (cat. no. 4511; Cell Signaling Technology, Inc.), JNK (cat. no. LF-PA0047; Thermo Fisher Scientific, Inc.), p-JNK (cat. no. sc-293136; Santa Cruz Biotechnology, Inc.), ERK (cat. no. 4695; Cell Signaling Technology, Inc.), p-ERK (cat. no. 4370; Cell Signaling Technology, Inc.), c-Jun (cat. no. sc-74543; Santa Cruz Biotechnology, Inc.), p-c-Jun (cat. no. sc-822; Santa Cruz Biotechnology, Inc.), c-Fos (cat. no. sc-7202; Santa Cruz Biotechnology, Inc.), p-c-Fos (cat. no. 5348s; Cell Signaling Technology, Inc.), β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) and lamin B1 (cat. no. sc-374015; Santa Cruz Biotechnology, Inc.).

Techniques: Irradiation, Incubation, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Glycine, the smallest amino acid, confers neuroprotection against d-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

doi: 10.1186/s12974-020-01989-w

Figure Lengend Snippet: Gly inhibited d -galactose-induced synaptic and memory dysfunction in C57BL/6 mice brain. a Western blot analysis of presynaptic proteins including synaptophysin (SYP), syntaxin (SYN), and postsynaptic density proteins (PSD95) in the hippocampus of different experimental groups The cropped bands were quantified using ImageJ software, and the differences are represented in the histogram. The density values are expressed in arbitrary units (A.U.) as the mean ± SEM for the respective indicated protein. An anti-β-actin antibody was used as a loading control. n = 8 mice/group, and the number of experiments performed N = 3. b The immunofluorescence images represented the immunoreactivity of PSD95 (green, FITC; blue, DAPI) in cortex and hippocampus of mice brain; along with their relative histograms, respectively. The relative integrated density values are represented in arbitrary units (A.U) as the means (± S.E.M) for the respective indicated proteins. DAPI (blue) was used for nucleus staining. n = 8 mice/group, and the number of experiments performed N = 3. Magnification × 40. Scale bar; 50 μm = cortices; DG hippocampal regions = 100 μm. c Mean escape latency in seconds to reach the hidden platform during training (5 days) along d with representative trajectories; f number of target crossings; g the time spent in the target quadrant; h Y-maze analysis represented spontaneous alteration behaviors along with its representative trajectories of mice. For behavioral study, the number of mice ( n = 16) per experimental group was used. Asterisk (*) sign indicated significant difference from the normal saline treated group; hash (#) sign indicated significant difference from d -gal-treated group; while the phi (Φ) sign indicated no significance from normal saline-treated group

Article Snippet: To confirm equal sample loading, an anti-β-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a standard for comparison.

Techniques: Mouse Assay, Western Blot, Software, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Glycine, the smallest amino acid, confers neuroprotection against d-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

doi: 10.1186/s12974-020-01989-w

Figure Lengend Snippet: Glycine treatment reduced d -galactose-mediated elevated p-JNK-dependent neuroapoptosis in HT22 cells lines. a Representative western blot analysis of activated phosphorylated (p-JNK), procaspase-3, BCL2-associated X protein (Bax), Bcl-2 (B-cell lymphoma 2), and poly [ADP-ribose] polymerase 1 (PARP-1) proteins expression levels with or without JNK inhibitor (SP600125) in the HT22 cell line. The cropped bands were quantified using ImageJ software, and the differences are represented in the histogram. The density values are expressed in arbitrary units (A.U.) as the mean ± SEM for the respective indicated protein. An anti-β-actin antibody was used as a loading control. Number of experiments performed N = 3. b , c Immunofluorescence images of activated p-JNK (green) and caspase-3 (red) proteins along with their relative histograms after drug treatment with d -gal (100 mM), Gly (20 μg/μl), and SP600125 (20 μM) treatment in HT22 cell line for 24 h. The relative integrated density values are represented in arbitrary units (A.U) as the means (± S.E.M) for the respective indicated proteins. DAPI (blue) was used for nucleus staining. The data are expressed as the mean ± SEM. Magnification × 40. Scale bar; 50 μm. a Significantly different from the control group while bcd significantly different from the d -gal-treated groups. Significance: a, b, c, d P

Article Snippet: To confirm equal sample loading, an anti-β-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a standard for comparison.

Techniques: Western Blot, Expressing, Software, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Glycine, the smallest amino acid, confers neuroprotection against d-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

doi: 10.1186/s12974-020-01989-w

Figure Lengend Snippet: Glycine treatment inhibited d -galactose-induced oxidative stress and ameliorates ROS/LPO production in mice brain. a , b Representative histograms of reactive oxygen species (ROS measured as DCF level) and lipid peroxidation (LPO measured as MDA level) assays of Gly against d -galactose in the cortex and hippocampus of mice brain. c – e Western blot analysis representing the expression level of Nrf2 and HO1 proteins in the hippocampus of the experimental groups. The cropped bands were quantified using ImageJ software, and the differences are represented in the histogram. The density values are expressed in arbitrary units (A.U.) as the mean ± SEM for the respective indicated protein. An anti-β-actin antibody was used as a loading control. n = 8 mice/group, and the number of experiments performed N = 3. g The immunofluorescence images represented the immunoreactivity of Nrf2 proteins (Red, TRITC; Blue, DAPI) in the cortex and hippocampus regions of mice brain along with their relative histograms, respectively. The relative integrated density values are represented in arbitrary units (A.U) as the means (± S.E.M) for the respective indicated proteins. DAPI (blue) was used for nucleus staining. n = 8 mice/group, and the number of experiments = 3. Magnification × 40. Scale bar; 50 μm = cortices; DG hippocampal regions =100 μm. Asterisk (*) sign indicated significant difference from the normal saline treated group; hash (#) sign indicated significant difference from d -gal-treated group; while the phi (Φ) sign indicated no significance from normal saline-treated control group. Significance: * P ≤ 0.05, ** P ≤ 0.01; *** P ≤ 0.001; # P ≤ 0.05, ## P ≤ 0.01

Article Snippet: To confirm equal sample loading, an anti-β-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a standard for comparison.

Techniques: Mouse Assay, Multiple Displacement Amplification, Western Blot, Expressing, Software, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Glycine, the smallest amino acid, confers neuroprotection against d-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

doi: 10.1186/s12974-020-01989-w

Figure Lengend Snippet: Gly inhibited d -galactose-induced activation of inflammatory proteins in the hippocampus of mice brain. a The Western blot analysis of tumor necrosis factor alpha (TNF-α), interleukin-1 βeta (IL-1βeta), glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule 1 (Iba1) protein expression level in the hippocampus of mice The cropped bands were quantified using ImageJ software, and the differences are represented in the histogram. The density values are expressed in arbitrary units (A.U.) as the mean ± SEM for the respective indicated protein. An anti-β-actin antibody was used as a loading control. n = 8 mice/group, and the number of experiments performed N = 3. b , c The immunofluorescence images represent the immunoreactivity of IL-1βeta (green, FITC; Blue, DAPI) in cortex and hippocampus of mice ( d ) The immunofluorescence images represent the immunoreactivity of GFAP (green, FITC; blue, DAPI) in cortex and hippocampus (CA-1 and DG regions) of mice The relative integrated density values are represented in arbitrary units (A.U) as the means (± S.E.M) for the respective indicated proteins. DAPI (blue) was used for nucleus staining. n = 8 mice/group, and the number of experiments = 3. Magnification × 40. Scale bar; 50 μm = cortices; DG hippocampal regions =100 μm. Asterisk (*) sign indicated significant difference from the normal saline-treated group; hash (#) sign indicated significant difference from d -gal-treated group; while the phi (Φ) sign indicated no significance from normal saline-treated control group. Significance: * P ≤ 0.05, ** P ≤ 0.01; *** P ≤ 0.001; # P ≤ 0.05, ## P ≤ 0.01.

Article Snippet: To confirm equal sample loading, an anti-β-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a standard for comparison.

Techniques: Activation Assay, Mouse Assay, Western Blot, Binding Assay, Expressing, Software, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Glycine, the smallest amino acid, confers neuroprotection against d-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

doi: 10.1186/s12974-020-01989-w

Figure Lengend Snippet: Glycine treatment inhibited d -galactose-induced elevated p-JNK and apoptotic cell death in mice brain. a Representative western blot analysis of stress kinase phosphorylated (p-JNK), cleaved caspase-3, cytochrome c (Cyt. C), Bcl-2 (B-cell lymphoma 2), and PARP-1 (poly-ADP-ribosyltransferase) proteins expression levels in both cortex and hippocampus regions of mice brain. The cropped bands were quantified using ImageJ software, and the differences are represented in the histogram. The density values are expressed in arbitrary units (A.U.) as the mean ± SEM for the respective indicated protein. An anti-β-actin antibody was used as a loading control. n = 8 mice/group, and the number of experiments performed N = 3. b Representative immunofluorescence results of caspase-3 (red; in hippocampus) and activated p-JNK proteins (green; in cortex and hippocampus) of the experimental mice group and c , d Nissl (cortex, DG, CA1, and CA3 regions) FJB staining (green, FITC; Blue) in cortex and hippocampus regions of experimental mice brain. The relative integrated density values are represented in arbitrary units (A.U) as the means (± S.E.M) for the respective indicated proteins. DAPI (blue) was used for nucleus staining. n = 8 mice/group, and the number of experiments = 3. Magnification × 40. Scale bar; 50 μm = cortices; DG hippocampal regions =100 μm. Asterisk (*) sign indicated significant difference from the normal saline treated group; hash (#) sign indicated significant difference from d -gal-treated group; while the phi (Φ) sign indicated no significance from normal saline-treated control group. Significance: * P ≤ 0.05, ** P ≤ 0.01; *** P ≤ 0.001; # P ≤ 0.05, ## P ≤ 0.01

Article Snippet: To confirm equal sample loading, an anti-β-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a standard for comparison.

Techniques: Mouse Assay, Western Blot, Expressing, Software, Immunofluorescence, Staining

Journal: Investigative and Clinical Urology

Article Title: Down-regulation of transient receptor potential melastatin member 7 prevents migration and invasion of renal cell carcinoma cells via inactivation of the Src and Akt pathway

doi: 10.4111/icu.2018.59.4.263

Figure Lengend Snippet: (A) Differentially expressed mRNA and protein levels of transient receptor potential melastatin member 7 (TRPM7) in various renal cell carcinoma lines. ACHN and SN12C cells were selected for gene silencing experiments because of the higher expression levels of TRPM7. (B) Small interfering RNA-mediated knockdown of TRPM7 in ACHN and SN12C cell lines analyzed by Western blotting. β-actin was used as an internal loading control. The band is a representative of three independent experiments. M, mock; N, negative control.

Article Snippet: An anti-β-actin antibody (#4967, rabbit polyclonal, 1:1,000; Cell Signaling Technology) was used as an internal loading control.

Techniques: Expressing, Small Interfering RNA, Western Blot, Negative Control

Journal: PLoS Biology

Article Title: A virus-acquired host cytokine controls systemic aging by antagonizing apoptosis

doi: 10.1371/journal.pbio.2005796

Figure Lengend Snippet: Secreted Diedel attenuates apoptosis in vitro. (A-C) Drosophila S2 cells exposed to UV (100 mJ/cm 2 ) and treated with mock or +Die V5 conditioned media (A) Dronc, (B) Puc, (C) Rpr transcription (measured by qRT-PCR); bars represent mean ± SE, n = 4. (D) Diedel transcription (measured by qRT-PCR) in S2 cells after exposure to UV, bars represent mean ± SE, n = 4. (E) dsRNA targeted against RFP (control) or Diedel (“Die”) in S2 cells exposed to UV (100 mJ/cm 2 ). Dronc transcription measured by qRT-PCR; bars represent mean ± SE, n = 3. (F-G) Immunoblot of protein extract from S2 cells exposed to UV (100 mJ/cm 2 ) and treated with Die V5 conditioned media at time 0 h, 1 h, 3 h, and 8 h, analyzed by western blotting with cleaved Cas-3 (“Cas 3 CL”) and normalized to beta-Actin (“beta-Act”). (G) Western blot quantification (normalized to beta-Actin), bars represent mean ± SE, n = 3. (H-I) Images of (H) cleaved Cas-3 (“Cas 3”) or (I) cleaved Dcp-1 (“Dcp1”) immunostaining in S2 Cells exposed to UV (100 mJ/cm 2 ) and treated with mock or Die V5 conditioned media (“CM”). (H) Cas-3 or (I) Dcp-1 (green), phalloidin (“Phall”; membrane, red), DAPI (blue). Bottom-right panel shows (5× zoom) Cas-3+ or Dcp-1+ cells in mock-conditioned media and UV-treated cells. Representative images shown. (J) Images of TUNEL immunostaining in S2 Cells exposed to UV (100 mJ/cm 2 ) and treated with mock or Die V5 conditioned media (“media”); TUNEL (nuclear, green), DAPI (blue), phalloidin (membrane, red). Bottom panels show TUNEL+ nuclei (white) with quantification; represents mean percent of TUNEL-positive nuclei (to total cells) ± SEM, n = 20–30. (K-L) Genomic DNA samples were isolated from S2 cells after UV exposition and treatment with (K) mock or Die V5 conditioned media (2 of 3 independent samples shown) or (L) purified Die V5 -tagged protein and separated by agarose gel electrophoresis. (M-N) Images of (M) cleaved Cas-3 (“Cas 3”) or (N) TUNEL immunostaining in MEFs exposed to UV (100 mJ/cm 2 ) and treated with mock or Die V5 Figs. Cas-3, caspase 3; Dcp-1, death caspase 1; +Die V5 , Diedel-V5; Dronc, death regulator Nedd2-like caspase; dsRNA, double-stranded RNA; MEF, mouse embryonic fibroblast; NS, not significant; Puc, Puckered; qRT-PCR, quantitative real-time PCR; RFP, red fluorescent protein; Rpr, Reaper.

Article Snippet: [DSHB Hybridoma Product UNC93-5.2.1]), and beta-Actin (Cell signaling, #4970).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Activated Clotting Time Assay, Immunostaining, TUNEL Assay, Isolation, Purification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Viruses

Article Title: N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle

doi: 10.3390/v12090925

Figure Lengend Snippet: The lack of N -glycan on the pre-membrane (prM) or/and E protein caused impairment in the E protein expression and secretion. ( a ) Confirmation of absence/presence of N -glycosylation in the prM- and E proteins expressed from constructed plasmids. For the Western blotting, proteins were stained with anti-ZIKV E -, anti-ZIKV prM- or anti-β actin protein antibody ( b ) Representative Western blot picture of E- and β-actin protein expression in harvested cell lysate and cell culture supernatant from three independent experiments. ( c ) Quantification of E protein expression with Image J. To normalize the E protein expression in the cell lysate, the expression level of E protein was divided by the expression level of β-actin. To normalize the E protein expression in the supernatant, the expression level of E protein from the supernatant was divided by the expression level of E protein from the cell lysate. The experiment was repeated three times, average and SD are shown. Statistical significance was determined by two-tailed t-test. p = p value. GFP = pcDNA3.1-GFP vector; WT clone = pcDNA3.1-prME vectors with wild-type ZIKV prM-E genes; N69Q = pcDNA3.1-prME vector with a mutated N -glycosylation site in prM; N154Q = pcDNA3.1-prME vector with a mutated N -glycosylation site in E; N69Q/N154Q = pcDNA3.1-prME vector with mutated N -glycosylation sites in prM and E; PNGase F = Peptide:N-glycosidase F.

Article Snippet: Proteins on the membranes were detected using rabbit anti-ZIKV E protein antibody or mouse anti-β actin (as an internal control for data analysis) antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted in 1x PBST with 5% nonfat dry milk.

Techniques: Expressing, Construct, Western Blot, Staining, Cell Culture, Two Tailed Test, Plasmid Preparation