Fig 2. IKK2-CA overexpressed HEK 293 cells were analyzed by PathwayScan Multi-Pathway Reporter Kit. 
 
                          
HEK 293 cells were cultured in 96-well plate in 100ul DMEM supplemented with 10% FCS and antibiotics during transfection and thereafter. Transfection were performed until the cells were 70% confluent. Transfection complexes were prepared by mixing reporter plasmids and lipofectamine 2000 in Opti-MEM. 0.1ug of one of 48 PathwayScan reporter plasmids (46 transcription factor reporter plasmids, a negative control plasmid and a positive control plasmid) was co-transfected with/without 0.1ug constitutive active IKK-beta (IKK2-CA, S177E, S181E) plasmid in each well.

The culture medium was then decanted 21 hours after transfection and 100ul of lysis buffer (10mM TrisHCl pH7.5 + 2mM DTT + 1% Triton X-100 + 2mM EDTA) was added. Luciferase activity of Firefly and Renilla in each well was determined by luminometer respectively. Firefly luciferase activity was then normalized to Renilla luciferase activity to reflect transcription factor activity.

To compare transcription factor activities between two groups (IKK2-CA overexpressed and non-overexpressed), the activities of two groups were first normalized to their negative control respectively, and an activity ratio of IKK2-CA overexpressed to non-overexpressed was calculated. The logarithm of the activity ratio with base 2 (Log2) was employed to show the activity difference. For a transcription factor, the value of Log2 is zero if its activity keeps intact; the value of Log2 is +4 if its activity increases 16 times; the value of Log2 is -2 if its activity decreases 4 times.

It is noted that IKK2-CA overexpression increased activity of transcription factor Erk (2.8-fold),NFkB (25-fold) as shown by green arrow in the figure, which are consistent with the literature that IKK2 activates Erk and NFkB activity. In addition, 5 transcription factors were found for the first time, whose activities were significantly changed by IKK2-CA overexpression (8.2-fold,12.1-fold, 3.8-fold increase and 7.7-fold, 6.3-fold decrease respectively, as shown by red arrow and labeled as New discovery).