Immunoblotting and Ubiquitination Analysis

As described previously, cells were lysed, sonicated and protein concentration was quantified by BCA analysis (Thermo Scientific, Rockford, IL). 50μg of protein lysate was resolved on SDS-PAGE, transferred to PVDF membrane, and blotted with specific primary and secondary antibodies (Sigma-Aldrich) accordingly. [15 (link)] For IP-Western, 500μg of protein lysate was incubated with Ubiquilin 1 tandem UBA (TUBE2) agarose (Boston Biochem, Cambridge, MA) in the IP buffer (25mM Tris•HCl, pH7.4, 150mM NaCl and 1% NP-40) on a rotation platform at 4ºC overnight. Ubiquitinated proteins were bound to the agarose, washed three times with IP buffer and subjected to immunoblotting. [55 (link), 56 (link)] Primary antibodies were used for Mcl-1, CDK9, actin (Santa Cruz Biotechnology, Dallas, TX), phospho-Pol II CTD at Ser2 and Ser5, Pol II CTD (Covance, Princeton, NJ), phospho-ERK1/2, ERK1/2, phospho-CDK9, phospho-Bcl-2, Bcl-xL (Cell Signaling Technology, Danvers, MA), Bcl-2 (Dako, Carpinteria, CA) and GAPDH (EMD Millipore, Billerica, MA). Densitometry of immunoreactive bands on immunoblots was measured with FluorChem E System (ProteinSimple, Santa Clara, CA), quantified and normalized with AlphaView (ProteinSimple).