puromycin resistance gene sequences  (TaKaRa)

Journal: The Journal of Clinical Investigation

Article Title: JAK2-V617F promotes venous thrombosis through β 1 /β 2 integrin activation

doi: 10.1172/JCI90312

Figure Lengend Snippet: Static adhesion on immobilized VCAM1 in the presence of (A) wortmannin (10 nM, 30 minutes) or (B) LY294002 (25 μM; 3 hours) in 32D JAK2-V617F and JAK2-WT cells. Minus symbol (–) indicates DMSO control. Representative Western blots (of 3 independent experiments) of phospho-Akt and Akt demonstrate the efficient blockade of signaling. (C) Inhibition of Rap1 activation by LY294002 treatment (25 μM; 3 hours) in 32D JAK2-V617F cells. Lower panel shows quantitative analysis of Rap1 activation given as fold compared with controls and corrected for loading variations using total Rap1; minus symbol (–) indicates DMSO control. (D) Rap1 pull-down and static adhesion on immobilized VCAM1 in the absence and presence of Akt inhibitor VIII treatment (0.5 μM; 4 hours) in 32D JAK2-V617F and JAK2-WT cells; minus symbol (–) indicates DMSO control. Representative Western blots (of 3 independent experiments) of phospho-Akt and Akt demonstrate the efficient blockade of signaling. (E) Static adhesion of 32D JAK2-V617F and JAK2-WT cells on immobilized VCAM1 in the absence and presence of BAPTA/AM (10 μM; 1 hour). (F) Static adhesion on immobilized VCAM1 of 32D JAK2-V617F cells transfected with scrambled (scr) or CalDAG-GEFI shRNA. (G) Rap1 pull-down after knockdown of CalDAG-GEFI results in reduced Rap1 activity. Data are shown as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (1-way ANOVA with Bonferroni’s post test and unpaired, 2-tailed Student’s t test). Three independent experiments were performed each.

Article Snippet: For knockdown of CalDAG-GEFI, the pLKO.1 vector system with a puromycin resistance gene containing one shRNA sequence for CalDAG-GEFI (Sigma-Aldrich) was used.

Techniques: Western Blot, Inhibition, Activation Assay, Transfection, shRNA, Activity Assay