Unless otherwise mentioned, all cell lines were obtained from American Type Culture Collection and used at early passage. Mutant p53 (R175H and R273H) overexpression plasmids encoding proline at codon 72 of p53 were a generous gift from Bert Vogelstein from Johns Hopkins School of Medicine. These plasmids were subjected to site-directed mutagenesis to generate R72 variants (Quick Change II, Stratagene), and all plasmids were verified by sequencing. Cells were transfected with the constructs of interest (5 µg of DNA) with Fugene and selected in 800 µg/mL neomycin for 14 d to make stable isogenic cell lines. For the luciferase plasmids, H1299 and PC3 cells were labeled with luciferase by lentiviral infection by using the plasmids pLenti-CMV-Puro-Luciferase (Addgene) and pLenti-UbC-RedFLuc-T2A-Puro (Targeting Systems, LP-30). The pLenti-UBc-RedFLuc-T2A-Puro plasmid was a generous gift from Dr. Meenhard Herlyn of The Wistar Institute. The antibodies used were as follows: p53 (DO-1) (Calbiochem, OP-43), GAPDH (Cell Signaling Technology, 2118S), TFAM (D5C8) (Cell Signaling Technology, 8076), PGC-1α (EMD Millipore, ST1202), PPARγ (R&D Systems, PP-A3409A-00), and Ki67 (D2H10) (Cell Signaling Technology, 9027).
Tfam d5c8
Manufactured by Cell Signaling Technology
TFAM (D5C8) is an antibody product from Cell Signaling Technology. It is a rabbit monoclonal antibody that recognizes the TFAM protein. TFAM is a transcription factor that regulates the expression of mitochondrial DNA.
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Lab products found in correlation
Plenti cmv puro luciferase, by Addgene (1 mentions)
Pgc 1α, by Merck Group (1 mentions)
Ki 67 d2h10, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Gapdh ga1r, by Thermo Fisher Scientific (1 mentions)
Cyclin b1 gns1, by SCBT (1 mentions)
Map lc3β g 9, by SCBT (1 mentions)
Opa1 612606, by BD (1 mentions)
Sdha d6j9m, by Cell Signaling Technology (1 mentions)
In cell analyzer 6000 cell imaging system, by GE Healthcare (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
3 protocols using tfam d5c8
1
Stable Isogenic Cell Lines from Mutant p53 Variants
2
Whole Cell Extract Preparation and Immunoblotting
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To prepare whole cell extracts from exponentially growing cultures, cells were lysed in RIPA lysis buffer [50 mM Tris, pH 8.0; 150 mM NaCl, 5 mM EDTA (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Pierce)]. Lysates were cleared by centrifugation at 12,000 rpm for 15 min and the supernatant was used for Western Blotting. Antibodies for immunoblotting were: TFAM [D5C8] (Cell Signaling), Drp1 [6Z-82] (SCBT), phospho-Drp1-Ser616 [D9A1] (Cell Signaling), cyclin B1 [GNS1] (SCBT), cyclin A [BF683] (SCBT), Ran [610340] (BD Biosciences), MAP LC3β [G-9] (SCBT), Mfn1 [D-10] (SCBT), GAPDH [GA1R] (Invitrogen), Opa1 [612606] (BD Biosciences), Nrf1 [147.1] (SCBT) and Nrf2 [A-10] (SCBT). Image quantification was performed in ImageJ (version 1.53a; freely available at http://imageJ.nih.gov/ij )90 (link). Full-size membranes of Western blots from which panels in Figs. 2–5 were prepared are shown in Supplemental Fig. S5 –S8 .
3
Mitochondrial Imaging and Profiling
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