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: International Journal of Molecular Sciences

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

doi: 10.3390/ijms19020598

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

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

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

Journal: International Journal of Molecular Sciences

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

doi: 10.3390/ijms19020598

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

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

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

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

Journal: Human Reproduction (Oxford, England)

Article Title: Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B

doi: 10.1093/humrep/dew295

Figure Lengend Snippet: Low oxygen tension modulates the effect of ET-1 on MMP14 and MMP15 down-regulation: Primary trophoblasts (GW 7–11, n = 12) were incubated under three different O 2 tensions (1% O 2 , 2.5% O 2 and 20% O 2 ) in the absence (control) or presence of 100 nM ET-1 for 24 h; ( A–C ) MMP14 (active form, act-MMP14) and MMP15 (pro-MMP15) protein levels were determined by Western blotting; ( D ) ET-1 levels were measured in the supernatants from the controls by ELISA; Results were normalized to β-actin protein levels (A and B) or total protein (D) and calculated as fold change relative to the controls (20% O 2 ), arbitrarily set to 1; Data are representative for at least four experiments; * P

Article Snippet: After blocking, the membranes were incubated with antibodies against MMP14 (Millipore, Billerica, MA, USA, 1:1500), MMP15 (Millipore, 1:500), HLA-G (BD-Biosciences, 1:1000), GAPDH (Novus, Littleton, CO, USA, 1:20 000) or β-actin (Abcam, Cambridge, MA, USA, 1:25 000) overnight at 4°C.

Techniques: Incubation, Activated Clotting Time Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Akt and Erk1/2 are likely the downstream targets of NONO-mediated signaling. p-Akt, Akt, p-Erk1/2 and Erk1/2 were compared between TE-1 and KYSE70 cell lines treated with control siRNA and NONO siRNA by western blot analysis. β-actin served as a loading control.

Article Snippet: The antibody against β-actin was purchased from Sigma-Aldrich.

Techniques: Western Blot

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Real-time PCR and western blot analysis of NONO expression in esophageal squamous cell carcinoma (ESCC) cells. (A) Real-time PCR measured relative NONO mRNA levels to GAPDH in 9 ESCC cell lines. (B) Western blot analysis of endogenous expression of NONO in all 9 ESCC cell lines. β-actin served as a loading control. (C and D) Transfection efficiencies of NONO siRNA knockdown in TE-1, KYSE70 cells were measured by real-time PCR and western blotting. (E) Immunofluorescence staining detected expression and nuclear localization of NONO protein in human ESCC cell lines. NONO expression is reduced after NONO siRNA knockdown in TE-1, KYSE70 cells.

Article Snippet: The antibody against β-actin was purchased from Sigma-Aldrich.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Immunofluorescence, Staining

Journal: International Journal of Molecular Medicine

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

doi: 10.3892/ijmm.2018.3414

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

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

Techniques: Expressing, Western Blot, Standard Deviation

Journal: BMC Cancer

Article Title: Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells

doi: 10.1186/s12885-018-4974-5

Figure Lengend Snippet: EOC-derived exosomes enhanced fibronectin and vitronectin expression in HPMCs through the transfer of miR-99a-5p. a Western blotting. HPMCs were transfected with miR-99a-5p or negative control miRNA. Thereafter, cell lysates were collected, and immunoblotting was performed with antibodies against fibronectin, vitronectin, or β-actin as a loading control. Representative blots from three independent experiments are shown. b After HPMCs were treated with IOSE- and EOC-derived exosomes at 100 μg/mL for 24 h, cell lysates were collected, and western blotting was performed as described above. c HPMCs transfected with negative control miRNA and anti-miR-99a-5p were treated with exosomes at 100 μg/mL for 24 h. Thereafter, cell lysates were collected, and western blotting was performed as described above. d Densitometric ratios of fibronectin, vitronectin, and β-actin expression. e Model. EOC-derived exosomes affect HPMCs through the transfer of miR-99a-5p. See text for details

Article Snippet: Antibody against β-actin (#4967) was purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Negative Control