A reporter plasmid carrying secreted alkaline phosphatase (SEAP) under the control of human CTGF promoter was a kind gift from Dr. Andrew Leask (Western University, Canada). Plasmids carrying Myc‐DDK tagged cDNAs for murine Yap (GeneBank accession# NM_001171147), and Hnf4α (NM_008261) were purchased (OriGene, Rockville, MD). These plasmids, empty pCMV6 vector or in combination (100 ng per well) were transfected into HEK293 or HepG2 cells in 24‐well plates. To knockdown human Hnf4α, 50 nM Stealth RNA oligonucleotides or non‐targeting scramble control was also transfected. One day after transfection, media were switched to conditioned media containing with or without Tgf‐β1 (5 ng/mL). A pCMV‐lacZ plasmid (Clontech) at 20 ng/well was transfected as internal control for normalization based on β‐galactosidase activities in the co‐transfected cells according to Leask et al.27 The SEAP activity was measured in conditioned medium 48 hours later using the Great EscAPe SEAP Chemiluminescence kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. SEAP activities were measured in triplicate experiments and relative CTGF promoter activity was expressed as fold change in comparison to the normalized SEAP activities of vector controls.
Pcmv lacz plasmid
Manufactured by Takara Bio
The PCMV-LacZ plasmid is a laboratory tool used for gene expression studies. It contains the lacZ gene, which encodes the enzyme beta-galactosidase, under the control of a cytomegalovirus (CMV) promoter. This plasmid can be used to monitor gene expression in various cell types.
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4 protocols using pcmv lacz plasmid
1
Investigating CTGF Promoter Activity Regulation
2
CTGF Promoter Activity Regulation by Yap and Hnf4α
3
TGF-β Signaling Pathway Activation
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LX-2 cells (2 × 105) were seeded in 24-well dishes and were transfected with 0.4 µg of the SBE4-Luc plasmid (luciferase reporter containing four copies of the Smad-binding site), and 0.1 µg of the pCMV-LacZ plasmid (Clontech) was co-transfected in all transfection experiments as an internal control. Cells were starved for 24 h followed by TGF-β (1 ng/ml) treatment for 24 h, and luciferase activities were measured with the Luciferase Assay system (Promega). All luciferase activities were normalized to β-galactosidase activity.
4
RNA FISH Probe Labeling and Detection
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RNA FISH probe labeling and RNA FISH procedures were performed as described previously [96 (link)]. Biotinylated single-strand LAT RNA probe was prepared by in vitro transcription (Ambion) using plasmid pSLAT-2 as a template (gift from S. Efstathiou, University of Cambridge, UK). Biotinylated LacZ probe was prepared from the pCMV-LacZ plasmid (Clontech) using the nick-translation procedure (Invitrogen). Frozen sections were treated as described for DNA FISH up to the antigen-unmasking step using solutions containing 2 mM of the RNAse inhibitor ribonucleoside vanadyl complex. The sections were pre-hybridized in 50% formamide/2 × SSC and hybridized overnight with 60 ng of RNA probe in a 50% formamide buffer at 65°C for LAT and 37°C for LacZ. Sections were washed in 50% formamide/2 × SSC at 65°C, and in 2 × SSC at room temperature. Detection was performed using streptavidin-HRP conjugate, followed by Tyramide Signal Amplification (TSA, Invitrogen) with an Alexa Fluor 350- or 488-conjugated substrate, according to the manufacturer’s guidelines. The DNA-FISH procedure was performed starting from the methanol/acetic acid post-fixation step.
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