Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Integrating serum exosomal microRNA and liver microRNA profiles disclose the function role of autophagy and mechanisms of Fructus Meliae Toosendan-induced hepatotoxicity in mice.

doi: 10.1016/j.biopha.2019.109709

Figure Lengend Snippet: Fig. 5. FMT disturbs the pathways predicted by IPA in mouse liver. (A) The expression levels of PTEN, PPP2R2A, p-PI3K, PI3K, p-AKT, AKT, and PUMA proteins in liver tissues of the mice exposure to high-dose FMT (n = 12 per group). (B) The expression levels of LC3-II, PINK1, and PARKIN proteins in liver tissues of the mice exposure to high-dose FMT (n = 12 per group). Protein samples with equal amount (100 μg) of twelve mice from the same group were first mixed together to be the sample for this group. The experiments were repeated three times. Data was shown as mean ± SD. The sig- nificance was determined by two-tailed un- paired Student's t test. * p < 0.05, ** p < 0.01, compared with control group.

Article Snippet: Then, membranes were blocked in TBST (Tris-buffered saline, 0.1 % Tween 20) containing 5 % non-fat dry milk or BSA followed by incubated with primary antibodies at 4°C against PPP2R2A (5689S, Cell Signaling, USA), PI3K (4292S, Cell Signaling, USA), p-PI3K (4228S, Cell Signaling, USA), AKT (4691S, Cell Signaling, USA), p-AKT (4060S, Cell Signaling, USA), p53 (2524S, Cell Signaling, USA), LC3(12741S, 4108S, Cell Signaling, USA), PUMA (AF1204, Beyotime Biotechnology, China), PTEN (AF1426, Beyotime Biotechnology, China), Bax (AF1270, Beyotime Biotechnology, China), GAPDH (AF0006, Beyotime Biotechnology, China), PINK1 (sc-33796, Santa Cruz Biotechnology, USA), and PARKIN (sc-30130, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Two Tailed Test, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammasome priming by LPS is dependent upon ERK signaling and proteasome function *

doi: 10.4049/jimmunol.1301974

Figure Lengend Snippet: Monocytes were nucleofected with ERK1 and ERK2 specific siRNA or scrambled control and recovered overnight. Cells were then primed with LPS (1μg/ml) for 30 min and activated with ATP (5mM) for another 30 min. Supernatants were analyzed for IL-18 release and cell lysates were immunoblotted to confirm ERK protein down regulation using total ERK antibody. IL-18 ELISA represents the mean± SEM for three separate monocyte donors and the blots are representative of repeated blots. **p < 0.01 as compared to scrambled siRNA.

Article Snippet: In knockdown experiments, small interfering RNA (siRNA) against ERK1 and scrambled siRNA were purchased from Sigma-Aldrich while Signalsilencer ® p42 MAPK (ERK2) siRNA II was purchased from Cell Signaling Company. siRNAs were delivered in monocytes by Amaxa nucleofector I (Lonza).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 2. Effect of HGF on p44/p42 MAPK (ERK-1/2) phosphorylation in lung fibroblasts from systemic sclerosis (SSc) patients. A, HGF-induced ERK-1/2 phosphorylation was determined in cells treated for various time periods with HGF (50 ng/ml). Cell extracts were immunoblotted with anti–phospho–ERK-1/2 (ph-ERK-1/2) or anti–ERK-1/2 (as a control) (see Materials and Methods). B, Dose-dependence of ERK-1/2 phosphorylation was determined by adding HGF at varying doses (5–500 ng/ml) to SSc lung fibroblasts for 10 minutes. C, Lung fibroblasts were transfected with or without Grb2 siRNA, MMP-1 siRNA, and control siRNA or pretreated for 40 minutes with or without MEK-1/2 inhibitor U0126 (1 M), MMP inhibitor GM1489 (10 nM), or NF-B inhibitor BAY 11-7082 (10 M), then incubated with HGF (50 ng/ml) for 10 minutes. Anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Quantitative results of densitometric analysis of immunoblots are also shown; values are the mean and SD from 3 independent experiments. P 0.05 versus unstimulated cells. See Figure 1 for other definitions.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Control, Transfection, Incubation, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot