1. Plate 500K cells/well for each conditon

Incubate o/n before transfection

For each transfection condition create the following tube mixtures. ( Volumes are for 1 transfection per condition and can be scaled up)

a. Tube 1: 125uL Optimem + 4uL lipofectamine 3000

b. Tube 2: 125uL Optimem + 2uL p3000 reagent + 1 ug total DNA

flick and incubate 5 minutes

Mix together tube 1 and tube 2 and incubate 15 min

Add dropwise to cells and incubate o/n

Incubate 8 hours

Change media to fresh media or media + 10mM EIPA

Incubate cells for 48 hours after transfection. Change media at 24 hours post transfection with fresh media and fresh media + EIPA

Slowly thaw luciferase reagent in room temp water

Wash gently each well with PBS

Add 500uL of luciferase reagent directly to plate and nutate for 10 min RT

Scrape each well with cell scraper and put into fresh microfuge tube

Spin down 13,000 rpm 5 min at RT

Add 100uL of supernatant to 4wells of white 96well plate. Wait 10 minutes

Read luciferase on plate reader

For Renilla reading, calculate reagent needed for 100uL each well using a 1:100 dilution of reagent: buffer

Add 100uL of renilla reagent to each well and wait 10 min before reading