β galactosidase assay kit

Forty-eight hours post-transfection, cells were harvested, lysed, and incubated in assay reagent according to the manufacturer’s instruction. Levels of active β-galactosidase expressed from BHK cells transfected with plasmids expressing lacZ gene were determined using the β-galactosidase assay kit according to the instructions of the manufacturer (Invitrogen, California, USA). The protein concentration of the cell lysate was determined using the Qubit^TM protein assay according to the manufacturer’s instructions (Invitrogen, California, USA). The specific activity of the cell lysate, determined in a total volume of 8 x 10⁵ nanolitres, was calculated as follows:
specific activity = nmoles of ONPG hydrolyzed/t/mg protein,
nmolesofONPGhydrolyzed=(OD420)(8x105nanolitres)4500nl/nmoles‐cm)(1cm)
where 4500 = the extinction coefficient, t = the time of incubation in minutes at 37°C, and mg protein = the amount of protein assayed.
For each experiment, the ratio of β-galactosidase activity of transfected BHK cells to that of the untransfected BHK cells was calculated. The ratios of relative β-galactosidase activities were compared using the independent samples t-test using SPSS version 25 (IBM Corp.).

RPMI and DMEM, DMEM F12 tissue culture media, Lipofectamine 2000 and β-galactosidase assay kit were purchased from Invitrogen; the luciferase Assay Reagent from Promega (Madison, WI); Troglitazone, TRAIL and Cycloheximide (CHX) were purchased from EMD Biosciences (Gibbstown, NJ), Compound C, AKT Inhibitor VIII, LY294002, Kenpaullone and AR-A014418 were from EMD Millipore (Billerica, MA), CHIR 99021 was from Sigma (St. Louis, MO). The antibodies utilized were obtained from the following sources: poly (ADP-ribose) polymerase (PARP), caspase-3, GSK-3β, phospho-GSK-3β^Ser9, GSK3α, AKT, pAKT^Ser473, pAKT2^Ser474, PPARγ, AMPKα1 and α2, GS, pGS^Ser641 from Cell Signaling Technologies (Danvers, MA); GAPDH from Ambion Inc. (Austin, TX). The GSK3β promoter luciferase plasmid (pGL3-GSK-3β-luc (−427/+66) was obtained from the laboratory of Dr. Daniel D. Billadeau, Mayo Clinic, Rochester, MN [34 (link)].

293T cells were transfected with PG13‐luc, a reporter gene containing the multiple copies of p53 consensus binding element linked to the luciferase gene with p53 and/or TAZ expression vector. Or cells were transfected with the p21‐promoter reporter gene (p21 pro‐luc) with p53 and/or TAZ expression vector. A pCMVβ vector was used as an internal control to compensate for the transfection efficiency. Reporter activity was assayed with a luciferase assay kit (Promega, Madison, WI, USA) or a β‐galactosidase assay kit (Thermo Fisher Scientific). Relative luciferase activity was determined after normalization to the β‐galactosidase activity and expressed as a fold induction.