β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 150µl of Assay 2X Buffer to sample. Incubate the reactions at 37°C for 24 hours	Read the absorbance at 420nm

Protocol tips
Add 150µl of Assay 2X Buffer to sample. Incubate the reactions at 37°C for 24 hours
Downstream tips
Read the absorbance at 420nm

Publication protocol

Smad3 Transcriptional Activity
Adherent cells were infected for 2h with adenovirus carrying a Smad3 reporter construct, (CAGA) 9MLP-Luc [14] or control adenovirus with β-Galactosidase [15]. After overnight starvation, TGF-β was added for 9h. Luciferase and β-Galactosidase activities were analyzed using Luciferase Assay Reagent and β-Galactosidase Enzyme Assay (Promega, Madison, WI, USA). After normalizing luciferase activity to β-Gal expression, TGF-β dependent induction of Smad3 transcriptional activity was normalized to the untreated control sample. Each condition was analyzed in triplicates.

Smad2 transcriptional activity
Smad2 transcription factor complexes recognize activin response elements (ARE). Smad2 is unable to bind DNA and needs assistance by, e.g. Fast-1, as cofactor. Hence, a luciferase gene under the control of ARE was co-transfected with a Fast-1 expression plasmid to evaluate Smad2/Smad4 transcriptional activity. For this, HCC cell lines were cultured at a confluency of 70-80%. ARE-Luc, Fast-1 and a β-Galactosidase control vector (pCR3lacZ; Invitrogen) at a ratio of 6:2:1 were introduced into the cell using Lipofectamine 2000 (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. After transfection, cells were starved over night until treatment with TGF-β for 9h. β-Galactosidase and Luciferase assay was performed as described above.

Smad7 Promoter Activity
β-Galactosidase control vector (pCR3lacZ; Invitrogen) and Smad7 promoter deletion mutant, p(-625 SacI)-Smad7prom-Luc, constructed from the 1,321-bp rat Smad7 promoter region (-1276 to -41) [16] were transiently transfected using Lipofectamine 2000 (Invitrogen, Darmstadt, Germany). Immediately post-transfection, cells were starved for 24h until treatment with TGF-β for 6h. β-Galactosidase and Luciferase assays were performed as described above

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