HEK293T cells were transfected with myc-tagged wild-type and mutant GFI1B cDNA using calcium phosphate method and maintained in DMEM medium (Euroclone, Pero, Italy) with 10% fetal bovine serum. Protein fractionated cell extracts were analyzed by western blot using primary antibodies anti-myc (9E10; sc-40; Santa Cruz Biotechnology, Dallas, TX, USA), anti-HSP90 (sc-7947; Santa Cruz Biotechnology), anti-ORC2 (ab68348; Abcam, Cambridge, UK); as previously described.23 (link)
Protocol detail
GFI1B Expression Analysis in HEK293T Cells
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Antibodies
Calcium phosphate
Cdna
Cell extracts
Cells
Fetal bovine serum
Hsp90
Orc2
Protein
Western blot
Corresponding Organization :
Other organizations : IRCCS Materno Infantile Burlo Garofolo, Royal North Shore Hospital, University of Sydney, University of Bologna, Policlinico San Matteo Fondazione, Istituti di Ricovero e Cura a Carattere Scientifico, University of Trieste
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Variable analysis
independent variables
- Wild-type GFI1B cDNA
- Mutant GFI1B cDNA
- Protein expression of myc-tagged GFI1B wild-type and mutant
- Protein expression of HSP90
- Protein expression of ORC2
- HEK293T cells
- Calcium phosphate transfection method
- DMEM medium with 10% fetal bovine serum
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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