Journal: Mediators of Inflammation

Article Title: Exosomes Play an Important Role in the Progression of Plasma Cell Mastitis via the PI3K-Akt-mTOR Signaling Pathway

doi: 10.1155/2019/4312016

Figure Lengend Snippet: AKT-mTOR signaling pathway activation was detected in PCM, and inhibiting exosome secretion reduced inflammation in vivo. (a) The expression of PIK3K, AKT, p-AKT, p-mTOR, HSP90AA1, and EEF2 was detected in PCM tissue sections by immunohistochemical staining. (b) Immunohistochemical staining scores were analyzed by the Mann-Whitney U test. There were no statistical differences for PIK3K and AKT in PCM tissues compared with NC tissues, while p-AKT and p-mTOR were overexpressed and HSP90AA1 and EEF2 were downexpressed in PCM tissues. ∗ p < 0.001 versus NC groups. (c) Western blots analyze changes of PI3K-Akt-mTOR pathway-related proteins. (d) Densitometric quantification for the changes of PI3K-Akt-mTOR pathway-related proteins was measured by QUANTITY ONE software. All values are expressed as the mean ± SD. ∗ p < 0.05 versus NC groups. (e, f) Pathological changes were detected by HE staining and inhibition of exosomes secretion by GW4869 significantly inhibited inflammatory cell infiltrate. Inflammatory infiltration scores were analyzed by the Mann-Whitney U test. ∗ p < 0.01.

Article Snippet: Samples were incubated with primarily indicated antibodies diluted at 1 : 100 overnight at 4°C: PI3K p85 alpha (Proteintech, 60225-1-Ig), p-AKT (Tr308) and AKT (pan) (Cell Signaling Technology), phospho-mTOR (Abcam, ab109268), HSP90AA1 (Boster, BA0369), and EEF2 (Boster, BM1733), and secondary rabbit-anti-human HistostainTM-SP Kit (SPN 9001 ZSGB-BIO) for 1 h at room temperature.

Techniques: Activation Assay, In Vivo, Expressing, Immunohistochemical staining, Staining, MANN-WHITNEY, Western Blot, Software, Inhibition

Journal:

Article Title: Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men

doi: 10.1113/jphysiol.2005.097154

Figure Lengend Snippet: eEF2 kinase was immunoprecipitated from human skeletal muscle samples and activity was measured in vitro in the absence and presence of Ca2+–calmodulin (Ca2+-CaM). Data are mean ±s.e.m. from 4 samples, *P < 0.01 versus EGTA.

Article Snippet: Briefly, eEF2 kinase was immunoprecipitated by incubating 3 μg of eEF2 kinase antibodies (US Biological, USA; E2213-50) with 20 μl of 33% (v/v) Protein-A-Sepharose (Amersham Biosciences, UK) and 50 μg of skeletal muscle lysate protein in 500 μl of immunoprecipitation buffer (IB; 10 m m Hepes, pH 7.2, 1 m m EGTA, 1 m m sodium pyrophosphate).

Techniques: Immunoprecipitation, Activity Assay, In Vitro

Journal:

Article Title: Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men

doi: 10.1113/jphysiol.2005.097154

Figure Lengend Snippet: Human skeletal muscle samples were subject to SDS-PAGE and immunoblotted with pT56-eEF2 (top panel) and eEF2 (bottom panel) antibodies. Data are mean ±s.e.m., n = 8; *P < 0.01 versus time 0. Representative immunoblots are shown. AU, arbitrary units.

Article Snippet: Briefly, eEF2 kinase was immunoprecipitated by incubating 3 μg of eEF2 kinase antibodies (US Biological, USA; E2213-50) with 20 μl of 33% (v/v) Protein-A-Sepharose (Amersham Biosciences, UK) and 50 μg of skeletal muscle lysate protein in 500 μl of immunoprecipitation buffer (IB; 10 m m Hepes, pH 7.2, 1 m m EGTA, 1 m m sodium pyrophosphate).

Techniques: SDS Page, Western Blot

Journal:

Article Title: Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men

doi: 10.1113/jphysiol.2005.097154

Figure Lengend Snippet: eEF2K was immunoprecipitated from skeletal muscle samples and in vitro activity was measured (top panel). Human skeletal muscle samples were subject to SDS-PAGE and immunoblotted with eEF2 kinase (bottom panel) antibodies. Data are mean ±s.e.m., n = 8; *P < 0.05 versus time 0. Representative immunoblots are shown. AU, arbitrary units; OD, optical density

Article Snippet: Briefly, eEF2 kinase was immunoprecipitated by incubating 3 μg of eEF2 kinase antibodies (US Biological, USA; E2213-50) with 20 μl of 33% (v/v) Protein-A-Sepharose (Amersham Biosciences, UK) and 50 μg of skeletal muscle lysate protein in 500 μl of immunoprecipitation buffer (IB; 10 m m Hepes, pH 7.2, 1 m m EGTA, 1 m m sodium pyrophosphate).

Techniques: Immunoprecipitation, In Vitro, Activity Assay, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: Inhibition of cytoplasmic cap methylation identifies 5′ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5′ ends

doi: 10.1093/nar/gkaa046

Figure Lengend Snippet: An inducible cell system to inactivate cytoplasmic cap methylation. A line of tetracycline-inducible U2OS cells was transfected with a plasmid bearing a tetracycline-regulated transgene expressing ΔN-RNMT. Clonal lines were selected by growth in medium containing G418 and analyzed for potential leakiness and inducible expression. One clonal line was selected for use in this study. ( A ) Cells carrying the ΔN-RNMT transgene were cultured for 24 h in the absence (–) or presence (+) of doxycycline (Dox), and cytoplasmic extracts were analyzed by western blotting with anti-RNMT antibody. The lower panel shows a portion of the same blot probed with antibody to EEF2 used in . ( B ) The same cell line grown on coverslips was again cultured for 24 h in the absence or presence of doxycycline, then fixed and stained with DAPI and antibody to the FLAG tag on ΔN-RNMT. The areas in white boxes are shown enlarged at the bottom of the figure.

Article Snippet: Membranes were blocked at room temperature in 3% BSA (w/v) in TBS for at least 30 min. Primary antibody staining was performed with rabbit anti-RNMT antibody (Proteintech 13743-1-AP, 1:500 dilution) or rabbit anti-EEF2 (One World Lab; 1:500 dilution) in 3% BSA (w/v) in TBS.

Techniques: Methylation, Transfection, Plasmid Preparation, Expressing, Cell Culture, Western Blot, Staining, FLAG-tag