Results Endogenous phosphorylated Akt accumulated

Endogenous phosphorylated Akt accumulated in the nuclei of HCC cells
Fig. 1.
Expression and cellular localization of endogenous Akt1 and phosphorylated Akt1 in HCC cells. (A) Immunocytochemistry for phospho-AktS473 (a,b), total Akt (c,d), phospho-FKHR (e,f) in SMMC-7721 Sotalol hydrochloride treated with or without wortmannin (10 ng/ml). (B) Phospho-AktS473 and total Akt in Huh-7 (a,c) and Hep3B (b,d) cells illustrated by immunocytochemical analysis. (C) Western blotting analysis of phospho-AktS473, phospho-AktT308, and total Akt from nuclear (nuc) and cytoplasmic (cyto) subfraction of SMMC-7721, Huh-7 and Hep3B HCC cell lines. The purity of the fractions was verified by anti-Lamin A/C and anti-tubulin antibodies. Original magnifications: A (a, b, c, d, e, and f), B (a, b, c, and d): 400×.
Figure optionsDownload full-size imageDownload as PowerPoint slide
Ectopic expression of GFP tagged Akt1 and its mutants distributes both in nucleus and cytoplasm of HCC cell
The cellular localization of GFP tagged Akt1 and its mutant Akt1-AA-GFP was Sotalol hydrochloride further studied. Following transfection with pEGFP-N1, Akt1-WT-GFP or Akt1-AA-GFP, SMMC-7721 cells were photographed by laser confocal scanning (LCS) microscope. As shown in Fig. 2A, Akt1-WT-GFP and Akt1-AA-GFP both distributed in the nucleus and cytoplasm. We further measured the fluorescence intensity in nucleus and cytoplasm of cells transfected with Akt1-WT-GFP or Akt1-AA-GFP, and the ratios of nuclear/cytoplasmic intensity were nearly equal (0.97 ± 0.11 and 0.97 ± 0.03, respectively). Both of them were little lower than that of GFP (1.02 ± 0.02) (Fig. 2B).

Antimicrobial peptides AMPs have broad spectrum

Antimicrobial iCRT 14 (AMPs) have broad-spectrum activity against bacteria, yeast, fungi, and virus, and play important roles in the defense system of many organisms from insects to humans [1]. AMPs have a low risk of resistance emergence and are being extensively proposed as new antimicrobial agents. One potential application of these peptides is the prevention and treatment of bacterial and fungal skin infections caused, for example, by Staphylococcal species and dermatophyte fungi. Particularly, mammalian skin and other epithelial surfaces are the major producers of functional cathelicidins [2] and [3]. The location and regulation of cathelicidin expression have indicated their importance in defence against skin pathogens [4] and [5]. The unique member of the cathelicidin family present in humans is hCAP18/LL37. Recently, it has been demonstrated that LL37 is produced by the eccrine apparatus and secreted into human sweat [6], where it is processed into multiple antimicrobial peptides, such as RK31 or KR20 [7].

Western blotting Cells were lysed in a TNT

Western blotting. Cells were lysed in a TNT buffer containing 20 mM Tris–HCl, DAPTinhibitor 7.5, 200 mM NaCl, 1% Triton X-100, 1 mM DTT and protease inhibitors (Roche, Basel, Switzerland). The protein content of the samples was measured using Pierce reagent following the manufacturer’s protocol. Protein samples of 20 μg were subjected to SDS polyacrylamide gel electrophoresis (PAGE) and the proteins were then electrophoretically transferred to a PVDF membrane using 100 V for 1 h at 4 °C. These membranes were then incubated with anti-NFAT, c-Fos, IκBα and β-actin antibodies at 1:500 dilutions in 5% dry milk solution plus 0.01% azide overnight at 4 °C. Subsequently, the blots were washed in TTBS (10 mM Tris–HCl, 50 mM NaCl, 0.25% Tween 20) and incubated with secondary antibody for 30 min at room temperature. The immunoreactive proteins were visualized using ECL (Amersham).
The number of TRAP-positive multinucleated osteoclasts decreases in co-cultures with mouse spleen cells and osteoblasts derived from TNFα and TNFRI deficient mice