Comprehensive Cell Culture and Antibody Protocol

HEK293T, HeLa and PINK1 KO³⁵ cells were cultured in Dulbecco's modified eagle medium (Life Technologies) supplemented with 10% (v/v) Fetal Bovine Serum (Gemini Bio Products), 10 mM HEPES (Life Technologies), 1 mM Sodium Pyruvate (Life Technologies), nonessential amino acids (Life Technologies) and GlutaMAX (Life Technologies). HeLa cells were acquired from the ATCC and authenticated by the Johns Hopkins GRCF Fragment Analysis Facility using STR profiling. All cells were tested for mycoplasma contamination bimonthly using the PlasmoTest kit (InvivoGen). Transfection reagents used were: Effectene (Qiagen), Lipofectamine LTX (Life Technologies), Avalanche-OMNI (EZ Bio-systems), X-tremeGENE HP (Roche) and X-tremeGENE 9 (Roche).
Rabbit monoclonal and polyclonal antibodies used: Beclin, pULK1-S317, pULK1-S757, TBK1, pTBK1-S172, NDP52, TAX1BP1, ATG5, Actin, and HA (Cell Signaling Technologies); GAPDH and LC3B (Sigma); ULK1 and Tom20 (Santa Cruz Biotechnology); Optineurin (OPTN) (Proteintech); GFP (Life Technologies); pSer65 ubiquitin (Millipore) and Mfn1 was generated previously^{36 (link)}. Mouse monoclonal antibodies used: NBR1 and p62 (Abnova), Cytochrome C oxidase subunit II (CoxII, Abcam), Parkin (Santa Cruz Biotechnology), DNA (Progen Biotechnik), ubiquitin (Cell Signaling). Chicken anti-GFP (Life Technologies) was also used. For catalog numbers see Supplementary Table 1. Human tissue panel blots were purchased (NOVUS Biologicals).

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Lazarou M., Sliter D.A., Kane L.A., Sarraf S.A., Wang C., Burman J.L., Sideris D.P., Fogel A.I, & Youle R.J. (2015). The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature, 524(7565), 309-314.

Publication 2015

Actin Amino acids Antibodies Avalanche Biologicals Cells Chicken Coxii Cultured medium Cytochrome c oxidase subunit ii Eagle Effectene Fetal bovine serum Gapdh Hela cells Hepes Human Lipofectamine Monoclonal antibodies Mouse Mycoplasma Novus Optineurin Parkin Progen Pyruvate Rabbit Sodium Tbk1 Tissue Transfection Ubiquitin Ulk1

Corresponding Organization :

Other organizations : National Institute of Neurological Disorders and Stroke, National Institutes of Health

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Protocol cited in 14 other protocols

Variable analysis

independent variables

Cell type (HEK293T, HeLa, PINK1 KO)
Transfection reagents (Effectene, Lipofectamine LTX, Avalanche-OMNI, X-tremeGENE HP, X-tremeGENE 9)

dependent variables

Protein expression and modification (Beclin, pULK1-S317, pULK1-S757, TBK1, pTBK1-S172, NDP52, TAX1BP1, ATG5, GAPDH, LC3B, ULK1, Tom20, Optineurin, GFP, pSer65 ubiquitin, Mfn1, NBR1, p62, Cytochrome C oxidase subunit II, Parkin, ubiquitin)

control variables

Culture medium (Dulbecco's modified eagle medium)
Supplements (10% Fetal Bovine Serum, 10 mM HEPES, 1 mM Sodium Pyruvate, nonessential amino acids, GlutaMAX)
Mycoplasma testing
STR profiling for HeLa cells

positive controls

None specified

negative controls

None specified

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