Immunoblotting Protein Detection Protocol

Immunoblotting to detect protein detection was performed using SDS-PAGE as previously described (Lal et al., 2014 (link)). Membranes for immunoblotting were probed with antibodies against anti-phospho-CTD-RNAPII-S2 (04-1571; Millipore), anti-phospho-CTD-RNAPII-S5 (04-1572; Millipore), anti-CDK7 (2916; Cell Signaling Technology), anti-CDK13 (A301-458A; Bethyl), anti-cleaved poly(ADP ribose) polymerase (Asp214; 9541; Cell Signaling Technology), anti-cleaved caspase 3 (Asp175; 9664; Cell Signaling Technology), NRF2 (12721; Cell Signaling Technology), and anti-α-tubulin (Sigma-Aldrich). Chemiluminescent (32106; Thermo Fisher Scientific) signal was captured using a Syngene G-BOX iChemi XT imager.

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Zarei M., Du H., Nassar A.H., Yan R.E., Giannikou K., Johnson S.H., Lam H.C., Henske E.P., Wang Y., Zhang T., Asara J, & Kwiatkowski D.J. (2019). Tumors with TSC mutations are sensitive to CDK7 inhibition through NRF2 and glutathione depletion. The Journal of Experimental Medicine, 216(11), 2635-2652.

Publication 2019

anti-CDK7 anti-cleaved caspase 3 (Asp175; 9664 anti-α-tubulin G-BOX iChemi XT imager

A301 Antibodies Caspase 3 Membranes Nrf2 Poly adp ribose polymerase Protein Rnapii Sds page α tubulin

Corresponding Organization : Harvard University

Other organizations : Texas A&M University, Dana-Farber Cancer Institute, Beth Israel Deaconess Medical Center

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Variable analysis

independent variables

Anti-phospho-CTD-RNAPII-S2 antibody
Anti-phospho-CTD-RNAPII-S5 antibody
Anti-CDK7 antibody
Anti-CDK13 antibody
Anti-cleaved poly(ADP ribose) polymerase (Asp214) antibody
Anti-cleaved caspase 3 (Asp175) antibody
NRF2 antibody
Anti-α-tubulin antibody

dependent variables

Protein detection

control variables

SDS-PAGE as previously described (Lal et al., 2014)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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