Immunoblotting of Mouse ESC Mitochondria

Immunoblotting was performed as previously described in Carbognin et al. (2016) (link). The following antibodies were used: anti-STAT3 mouse monoclonal (9139, Cell Signaling Technology, 1:1000), anti-GAPDH mouse monoclonal (MAB374, Millipore, 1:1000), anti-VDAC1 rabbit polyclonal (ab15895, Abcam, 1:1000), anti-Lamin (sc-6217, Santa Cruz Biotechnology, 1:1000), anti-βActin mouse monoclonal (MA1-744, Invitrogen, 1:10,000) and anti-pSTAT3 S727 rabbit monoclonal (9134, Cell Signaling Technology). Mitochondria from mouse ESCs were isolated using Mitochondria Isolation Kit (89874, Thermo Fisher Scientific).

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Peron M., Dinarello A., Meneghetti G., Martorano L., Betto R.M., Facchinello N., Tesoriere A., Tiso N., Martello G, & Argenton F. (2021). Y705 and S727 are required for the mitochondrial import and transcriptional activities of STAT3, and for regulation of stem cell proliferation. Development (Cambridge, England), 148(17), dev199477.

Publication 2021

anti-STAT3 mouse monoclonal anti-GAPDH mouse monoclonal ab15895 Mitochondria Isolation Kit

Antibodies Escs Gapdh Isolation Lamin Mitochondria Mouse Rabbit Stat3 Vdac1

Corresponding Organization :

Other organizations : University of Padua

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

STAT3 protein levels
GAPDH protein levels
VDAC1 protein levels
Lamin protein levels
βActin protein levels
PSTAT3 S727 protein levels

control variables

Immunoblotting protocol described in Carbognin et al. (2016)
Antibody concentrations used (1:1000 or 1:10,000)
Mitochondria isolation method using Mitochondria Isolation Kit (89874, Thermo Fisher Scientific)

controls

Positive control: None explicitly mentioned
Negative control: None explicitly mentioned

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