control cmv tgfp  (OriGene)

Journal: Molecular carcinogenesis

Article Title: The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation

doi: 10.1002/mc.22636

Figure Lengend Snippet: The effect of PHB knockdown on Akt kinase activity as well as cell survival and proliferation upon UVB irradiation. HaCaT cells were stably transfected with scrambled shRNA (control) or PHB shRNA (PHB knockdown). (a) PHB expression in the cells was examined by western blotting analysis. The data represents three experiments. (b) Phospho-Akt in the cells treated or not treated with UVB (50 mJ/cm2, 6 h) was immunoprecipitated by immoblilized p-Akt primary antibody and incubated with GSK 3 fusion protein. Then the phosphorylation of GSK-3α/β was detected by western blotting analysis. The data represents three experiments. (c) Survival and proliferation of the cells treated or not treated with UVB (5 mJ/cm2) were assessed by clonogenic assay. The data represents the means ± SD of three independent experiments.

Article Snippet: HaCaT cells were transfected with pLKO.1-Neo-CMV-tGFP vectors containing PHB shRNA or scrambled control shRNA (Sigma, St. Louis, MO) using Lipofectamine LTX and PLUS following the manufacture’s protocol (Invitrogen, Carlsbad, CA).

Techniques: Activity Assay, Irradiation, Stable Transfection, Transfection, shRNA, Expressing, Western Blot, Immunoprecipitation, Incubation, Clonogenic Assay

Journal: Molecular carcinogenesis

Article Title: The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation

doi: 10.1002/mc.22636

Figure Lengend Snippet: The role of PHB in UVB-induced apoptosis in HaCaT cells. (a) HaCaT cells were transiently transfected with scrambled siRNA or PHB siRNA. The cells without transfection were used as control. The levels of PHB, p-ERK, ERK, p-Akt, Akt, and β-actin in the transfected cells with or without UVB (50 mJ/cm2, 6 h) were assessed by western blotting analysis. The data represents three experiments. (b) The levels of p-Akt, Akt, caspase-3, and β-actin in Akt I (1 μM) and/ or UVB (50 mJ/cm2, 6 h) treated cells were assessed by western blotting analysis. The data represents three experiments.

Article Snippet: HaCaT cells were transfected with pLKO.1-Neo-CMV-tGFP vectors containing PHB shRNA or scrambled control shRNA (Sigma, St. Louis, MO) using Lipofectamine LTX and PLUS following the manufacture’s protocol (Invitrogen, Carlsbad, CA).

Techniques: Transfection, Western Blot