Journal: Scientific Reports

Article Title: Protein malnutrition potentiates the amplifying pathway of insulin secretion in adult obese mice

doi: 10.1038/srep33464

Figure Lengend Snippet: GDH modulates amplifying pathway of insulin secretion in islets from malnourished obese mice: mRNA expression for GLUD1/GAPDH ( A ). Protein content for GDH ( B ). NADH concentration ( C ). GDH activity ( D ) Pancreatic islets were isolated and incubated with glucose 22.2 mM in the presence of Dz (250 μM) and K + (30 mM) with or without addition of epigallocatechin gallate (EGCG) (20 μM). The value Δ was obtained by subtracting of secretion with Glucose + K + + Dz less insulin secretion with G22.2 ( E ). Protein content for murf 1 ( E ), proteasome β subunit ( F ) and ubiquitin ( G ). Overview of insulin secretion in β-cells mediated by GDH ( I ). Results are means ± SEM (n = 4–9) (p < 0.05) Ω R, in ANOVA Newman Keuls´s post-test or & as represented in Student t test.

Article Snippet: For Western blotting, 20 μg of the total protein for PKAα (#903; SCBT), PKCα (#8393; SCBT), cleaved caspase-3 (#9662; Cell Signaling), UCP2 (#77363; Abcam), GDH (#400051; US-Biological), Murf1 (#32920; SCBT), Proteasome subunit β type 2 (#22650; Abcam), Ubiquitin (#7780; Abcam) and the housekeeping proteins GAPDH (#3873; Cell Signaling Technology) and HSP90 (#101494; SCBT) were resolved using 10% SDS-PAGE and electroblotted onto nitrocellulose membranes.

Techniques: Expressing, Concentration Assay, Activity Assay, Isolation, Incubation, Ubiquitin Proteomics

Journal: International journal of cancer

Article Title: MUTYH c.933+3A>C, associated with a severely impaired gene expression, is the first Italian founder mutation in MUTYH-Associated Polyposis.

doi: 10.1002/ijc.27761

Figure Lengend Snippet: Figure 3. Quantitation of MUTYH gene and protein expression in LCLs (a) mRNA expression analysis by real-time PCR. Graphs report the mean ratio of MUTYH exon 5–6, 9–10 and 9–11 assay signals normalized by b-tubulin and b-actin genes (mean of three experiments with standard deviations). Control bars are WT (mean results obtained on two wild-type LCLs) for the 5–6 and 9–10 assays, FAP349 (c.933þ3A>C homozygote) for the 9–11 assay. An assay scheme is reported below each graph (P, probe; FW, forward primer; REV, reverse primer). *p < 0.05 and **p < 0.01 relative to controls. (b) Western blot analysis of MUTYH protein. Bars with standard deviations represent the mean ratio between MUTYH and b-tubulin for three experiments on each sample. **p < 0.01 and ***p < 0.001 relative to WT controls. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: MUTYH signals were normalized using the b-tubulin housekeeping protein (rabbit polyclonal anti-tubulin b, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Quantitation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Scientific Reports

Article Title: Prototheca zopfii genotype II induces mitochondrial apoptosis in models of bovine mastitis

doi: 10.1038/s41598-020-57645-z

Figure Lengend Snippet: Prototheca zopfii genotype II-induced microscopic changes in the mammary gland of mice. ( A ) H&E, PAS and GMS staining and immune detection of macrophages (F4/80) and neutrophils (Ly6 G) in PBS control and P. zopfii -infected mammary tissues at 4 d post infection. Note infiltration of macrophages and neutrophils and presence of innumerable P. zopfii GT-II as detected by PAS and GMS staining. Bar = 20 µm. ( B ) Transcriptomic expression of genes of pro-inflammatory TNF- α, IL-1β and Cxcl-1 after infection with P. zopfii GT-II. ( C ) ELISA titers of TNF-α and Cxcl-1 in mammary tissue. ( D ) Quantitative detection of apoptotic cells in PBS control and P. zopfii GT-II infected mammary tissue in mouse mammary gland (green signal indicates TUNEL apoptotic cells). ( E ) Transcriptomic expression of Bcl-2 , cytochrome-c, caspase-9, and caspase-3 in mouse mammary tissues infected with P. zopfii GT-II. ( F ) Proteomic expression of activated caspase-3 in mammary gland 4 d after P. zopfii GT-II infection, tubulin and activated caspase-3 run on different gels at same time and cropped according to respective size. * P < 0.05, ** P < 0.01.

Article Snippet: Membrane was blocked with 5% skim milk in TBS-T (150 mM NaCl, 10 mM Tris base, 0.05% Tween 20, pH 7.4) at room temperature for 120 min and then incubated overnight at 4 °C with primary antibodies for caspase-9 (Cat # ab69514, Abcam USA), caspase-3 (Cat # ab90437, Abcam USA), cytochrome-c (Cat # ab110325, Abcam USA) and housekeeping β-tubulin (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Control, Infection, Expressing, Enzyme-linked Immunosorbent Assay, TUNEL Assay