Journal: Journal of leukocyte biology

Article Title: N6-isopentenyladenosine, an endogenous isoprenoid end product, directly affects cytotoxic and regulatory functions of human NK cells through FDPS modulation.

doi: 10.1189/jlb.0413190

Figure Lengend Snippet: Figure 5. Molecular analysis of the iPA-mediated effects on NK cells functions: evidence for an exclusive modulation of MAPK. (A) Puri- fied NK cells were treated with IL-2 alone or in combination with the indicated concentrations of iPA for 18 h (overnight). Representative blots (left) and quantitative/densitometric analysis (arbitrary units; right) of results from four different experiments are shown. All of the protein levels were calculated by normalizing the levels of p-ERK1/2, total ERK1/2 proteins, p-p38, total p38 protein, p-STAT5, and total STAT5 protein with the levels of -actin, which serves as internal load- ing control. The relative levels of specific p-ERK, p-p38, and p-STAT5 were then calculated by normalizing the levels of phosphorylated forms with the levels of their cognate proteins. All four experiments were performed in duplicate with similar results (ANOVA; *P0.05; **P0.01). (B) Representative WB (upper) and quantitative/densito- metric analysis (arbitrary units; lower) of results from three different experiments conducted on purified NK cells, stimulated with IL-2 alone or in combination with the indicated concentrations of iPA at different time-points from 30 min to 3 h are shown. All three experi- ments were performed with similar results (ANOVA; *P0.05; **P0.01).

Article Snippet: The following antibodies were used: mouse monoclonal anti-human panRas was purchased from Sigma-Aldrich or Cytoskeleton; rabbit monoclonal anti-human p-STAT5 (Tyr 694), rabbit polyclonal anti-human STAT5, rabbit monoclonal anti-human p-p44/42 MAPK (p-ERK, Thr202/Tyr 204), rabbit monoclonal anti-human p44/42 MAPK, mouse monoclonal anti-human p-p38 MAPK (Thr180/Tyr182), and rabbit monoclonal anti-human p38 MAPK were purchased from Cell Signaling Technology; rabbit polyclonal anti-human -actin and rabbit polyoclonal anti-human FDPS were purchased from Abcam (Cambridge, UK).

Techniques: Control

Journal: Cancer Research

Article Title: Proapoptotic Activity of New Glutathione S-Transferase Inhibitors

doi: 10.1158/0008-5472.can-04-3903

Figure Lengend Snippet: Figure 6. p38MAPK pathway is involved in NBDHEX-induced apoptosis. A, CCRF-CEM and K562 cell lines were treated with 2 and 10 Amol/L NBDHEX, respectively, up to 24 hours. At the indicated time, samples (20 Ag of cell extracts) were obtained and subjected to immunoblotting analysis to detect phosphoactivated forms of p-38 (P-p38MAPK). h-Actin was used as loading control. B, K562 cells were preincubated with 10 Amol/L SB203580 (specific p38MAPK inhibitor) before the addition of 10 Amol/L NBDHEX. At different times, aliquots of cell suspension were withdrawn and treated for Western blot analysis to detect p-38MAPK and its phosphoactivated form, P-p38MAPK. C, 10 Amol/L SB203580 were added before the NBDHEX treatment and maintained throughout. At the indicate time, the percentage of apoptosis in CCRF-CEM and K562 cells was determined by fluorescence microscopy after staining with Hoechst. Columns, means (n = 3); bars FSD. *, P < 0.05.

Article Snippet: Polyclonal anti-total (Santa Cruz Biotechnology), phosphoisoform of p38MAPK (Cell Signaling Technology, New England Biolabs, Cancer Res 2005; 65: (9).

Techniques: Western Blot, Control, Suspension, Fluorescence, Microscopy, Staining