Luminescent β-galactosidase Detection Kit II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature	Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Upstream tips
Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature
Protocol tips
Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Publication protocol

The promoter region of human (−419/−1), (−1548/+63), (−1343/+62), and (−588/+155) genes was amplified by PCR and subcloned into pGL3-basic (Promega). The CArG mutant constructs were designed with site-directed mutation in the CArG boxes (CC[A/T]6GG [wild type] to AA(A/T)6GG or CC(A/T)6AA [mutant]). The 3× CArG-Luc vector was constructed as reported previously (). These constructs were introduced into HeLa or A7r5 cells along with pSV-βGal (Promega), which was used to normalize the transfection efficiency. 2 d after transfection, luciferase and β-galactosidase activities were measured using the Luciferase Assay System (Promega) and Luminescent β-Galactosidase Detection Kit II (Takara Bio Inc.), respectively. For the reporter assay in Arp5-knockdown cells, A7r5 cells were transfected with reporter constructs for 24 h and then transfected with Arp5 siRNA2. 5 d after siRNA transfection, luciferase and β-galactosidase activities were measured.

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