Journal: Journal of Translational Medicine

Article Title: Inhibition of mitotic kinase Aurora suppresses Akt-1 activation and induces apoptotic cell death in all-trans retinoid acid-resistant acute promyelocytic leukemia cells

doi: 10.1186/1479-5876-9-74

Figure Lengend Snippet: VX-680 induces mitochondrial depolarization and cellular caspase activation in NB4-R2 cells . (A) VX-680 treated NB4-R2 cells were stained with JC-1 probe and measured by flow cytometry. X- and Y-axes were indicative of monomer and aggregates, respectively. Data shown is a representative of three independent experiments. (B) NB4-R2 cells were collected, lysed and subjected to Western blot analysis with cleaved caspase-3, cleaved-PARP, pAkt-1 (Ser473), pGSK-3β (Ser9) specific antibodies. GAPDH was used as a loading control. Data shown is a representative of three independent experiments.

Article Snippet: The membrane was blocked and then incubated with GAPDH (from Ambion), p-Aur-A/AIK (Thr288), cleaved PARP (Asp214), pAkt-1 (Ser473), cleaved caspase-3 (Asp175) and pGSK-3 (Ser9) antibodies (from Cell Signaling), at 4°C overnight, followed by incubation for 1 hr RT with appropriate secondary antibodies.

Techniques: Activation Assay, Staining, Flow Cytometry, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Maladaptive Modulations of NLRP3 Inflammasome and Cardioprotective Pathways Are Involved in Diet-Induced Exacerbation of Myocardial Ischemia/Reperfusion Injury in Mice

doi: 10.1155/2016/3480637

Figure Lengend Snippet: Prosurvival RISK pathway activation in the mouse heart. Representative western blotting for cardiac levels of total ERK1/2 expression and Thr202/Tyr204 phosphorylation, respectively, (a), total Akt protein expression and Ser473 phosphorylation (b), and total GSK-3 protein expression and Ser9 phosphorylation (c) performed on heart apex extracts from Control or HFHF diet mice, exposed or not to IR. Histograms report densitometric analysis of the phosphorylated-to-total form ratio of 5-6 mice per group. ∗ P < 0.05 versus Control; • P < 0.05 versus Sham.

Article Snippet: Membranes were probed with goat anti- α -myosin heavy chain ( α -MHC), goat anti- β -MHC, rabbit anti-carnitine palmitoyltransferase (CPT) 1m, mouse anti-succinate dehydrogenase (SDH), anti-glucose transport- (GLUT-) 4 primary antibody (Abcam, Cambridge, UK), rabbit anti-phospho-insulin receptor 2 (IRS2 Ser270 ), and mouse anti-IRS-2 (Cell Signaling, Danvers, MA, USA), rabbit anti-Nlrp3 (Epitomics, Burlingame, CA, USA), rabbit anti-caspase-1, rabbit anti-pERK1/2, rabbit anti-ERK, rabbit anti-pAkt Ser473 , rabbit anti-Akt, rabbit anti-pGSK-3 β Ser9 , rabbit anti-GSK-3 β , mouse anti-hypoxia inducible factor- (HIF-) 2 α , and goat anti-hydroxynonenal (HNE) (Novus Biologicals, Abingdon, UK) primary antibodies, followed by incubation with appropriated HRP-conjugated secondary antibodies (Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Activation Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury

doi: 10.3390/ijms222111768

Figure Lengend Snippet: The effect of hydrogen peroxide, insulin and α-T on the activity of protein kinase GSK-3beta [pGSK-3beta (Ser 9 )/GSK-3beta] in brain cortical neurons. ( A ) immunoblots of pGSK-3beta (Ser 9 ) and total GSK-3beta are shown; ( B ) the data of 10 experiments are presented as the means ± SEM. The brain cortical neurons were pre-incubated for 1 h with 1 μM insulin or for 18 h with 50 μM α-T or with both drugs or without them. Then the neurons were exposed to 100 μM hydrogen peroxide for 0, 15 and 360 min. The differences are significant according to Student’s paired t -test as compared: * and **—to the control, * p < 0.02, ** p < 0.05; x—to the effect of hydrogen peroxide alone, p < 0.01, #—to the effect of insulin or α-T alone, p < 0.01.

Article Snippet: Monoclonal antibodies specific for pAkt (Ser 473 ) (1:1000, #4058, Cell Signaling Technology, Danvers, MA, USA), for pGSK-3beta (Ser 9 ) (1:1000, #9322, Cell Signaling Technology), for pERK1 (pThr 202 /pTyr 204 ) and pERK2 (pThr 185 /pTyr 187 ) (1:2000, #E7028, Sigma-Aldrich), and for cleaved caspase-3 (Asp175) (1:1000, #9664, Cell Signaling Technology) were used to probe the blots overnight at +4 °C.

Techniques: Activity Assay, Western Blot, Incubation