Journal: Biological Research
Article Title: CCAT2 is an oncogenic long non-coding RNA in pancreatic ductal adenocarcinoma
doi: 10.1186/s40659-017-0149-0
Figure Lengend Snippet: KRAS regulates CCAT2 expression via MEK/ERK pathway. a The expression of KRAS was compared between Scramble- and si-KRAS-treated PANC-1 cells 48 h after transfection. b CCAT2 expression in PANC-1 cells transfected with si-KRAS or Scramble. *P < 0.05. c Immunoblotting for p-ERK, total ERK, p-AKT and total AKT in si-KRAS-treated and Scramble-treated PANC-1 cells 48 h after transfection. d PANC-1 cells were co-transfected with CCAT2 promoter-reporter plasmid in combination with si-KRAS or Scramble. Luciferase activity were measured 48 h after transfection. *P < 0.05. For a and c , GAPDH was used as loading control. Image shown was representative of three independent biological repeats. For b and d , data shown were mean ± SEM of three independent biological repeats. e Activation of ERK or AKT was evaluated in PANC-1 cells treated with MEK/ERK inhibitor PD98059 or PI3K/AKT inhibitor LY294002 for 24 h. f CCAT2 expression in PANC-1 cells treated with PD98059 or LY294002 for 24 h. *P < 0.05. For e and f , DMSO-treated group was used as control. Image shown was representative of three independent biological repeats
Article Snippet: Primary rabbit antibodies against total ERK (No. 4695), p-ERK (No. 4370), total AKT (No. 4685), p-AKT (No. 4060), KRAS (No. 3339) (Cell Signaling Technology Inc., Berkeley, CA, USA) and GAPDH (No. TA-08) (loading control) (Zhongshanjinqiao Biotech, China) were incubated at 4 °C overnight at a dilution of 1:1000, and after washed with PBST for three times, the secondary horseradish-peroxidase-labeled antibody was incubated at room temperature for 2 h at a dilution of 1:5000.
Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Luciferase, Activity Assay, Control, Activation Assay
