1) Objective：
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a unique adaptor protein of the tumor necrosis factor receptor-associated factor. In the research we want to demonstrate the over-expression of TRAF6 might be related to the tumo-rigenesis and invasion of lung cancer
2) Method:
a) Cell culture
b) Western blot
Separated by SDS-PAGE Transferred to a PVDF membrane Blocked for 1 h in TBS solution with 5 % skimmed milk,probed with primary antibody at 4℃ overnight probed with corresponding secondary antibody at room temperature for 2 h Use the enhanced chenmiluminescerce detectionsystem to detect the blot
c) Knockdown of TRAF6 expression with short-hairpin RNA
d) Determination of cell viability
Seed cells into 96-well plates and allowed to adhere overnight Add MTT into the plate and incubate for 4-6h Media were then removed, and add 150 μL DMSO Micro-plate Reader
e) Determination of cell cycle by flow cytometry
f) Determination of invasion capability of lung cancer cells
3) Result:
a) It is concluded that TRAF6 might be closely associated with tumorigenesis, metastasis, and invasion.
b) We supposed that inhibition of TRAF6 attenuated tumor cell proliferation and invasion and promoted apoptosis possibly through downregulation of p-p65, thereby affecting the expression of cyclin D1, caspase-3, and MMP9 in A549 cell line. |