Protocol tips
Upstream tips |
Protocol tips |
Downstream tips |
Seed 1 × 10^5 cells/well |
Add 2 ml of 1X Fixing Buffer and incubate 5–10 minutes at room temperature.
Add 2 ml of the X-Gal Staining Mix to the dish and incubate the dish at 37°C for 1 hr to overnight |
|
Upstream tips |
Seed 1 × 10^5 cells/well |
Protocol tips |
Add 2 ml of 1X Fixing Buffer and incubate 5–10 minutes at room temperature.
Add 2 ml of the X-Gal Staining Mix to the dish and incubate the dish at 37°C for 1 hr to overnight |
Publication protocol
Cells were plated in 12-well dishes at 1 × 105 cells/well and 24 h later transfected in Opti-MEM (Invitrogen) with pCMV-β-galactosidase and the indicated plasmids using either Lipofectamine 2000 (Invitrogen) for HEK293T cells or Lipofectamine LTX with PLUS reagent for INS1 cells following the manufacturer’s protocol. After 24 h, cells were harvested and luciferase and β-galactosidase activities measured using a luciferase assay kit (Promega) and a luminometric β-galactosidase detection kit (Clontech), respectively, following the manufacturer's protocols. Each data point was assayed in triplicate and each experiment was performed at least twice. Relative luciferase activity was calculated. All values underwent analysis of variance and Tukey-Kramer comparison tests using InStat software (GraphPad Software Inc.), and data are presented as mean ± S.E.
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HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity
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