Hrp conjugated actin

The mitochondrial extracts were prepared as described (19 (link)). Protein concentration was determined with Bradford reagent (Bio-Rad, Hercules, CA, USA). Western analysis was performed as described (21 (link)) with the membranes sequentially probed using antibodies against PARP1, HRP-conjugated actin (Santa Cruz, Dallas TX, USA), FEN1, APE1, Polγcat, Polγacc, Lig3, Polβ (Gene Tex, Irvine, CA, USA), PAR (BD Pharmingen, San Jose, CA, USA), GAPDH (Cell Signaling, Danvers, MA, USA), the 56 kDa subunit of ATP synthase (Molecular Probe-Invitrogen, Carlsbad, CA, USA), FeS subunit of mitochondrial complex III, subunit of complex I (NDUFS3), (Abcam, Cambridge, MA, USA), EXOG and FLAG-HRP conjugated (Sigma).

For protein purification, cells were mechanically harvested in Western blot PRO-PREP lysis buffer (Intron Biotechnology) and homogenized with Vibra cell^TM VCX-750 (SONICS & MATERIALS Inc., Newton, CT) if required. The proteins were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The protein-bound membranes were blocked with 4% BSA in TBST for 1 h at RT followed by overnight incubation with a primary antibody at 4°C. The primary antibodies used in this study were as follows: ZEB1 (1:200, Santa Cruz Biotechnology), E-CADHERIN (1:1000, BD Biosciences), and HRP-conjugated actin (1:1000, Santa Cruz Biotechnology). After washing several times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Santa Cruz Biotechnology). The membranes were developed with ECL (Elpis biotech) and exposed on a Fujifilm LAS-4000 CCD camera system (Fujifilm, Tokyo, Japan) for quantitative imaging. Band intensities were measured with the ImageJ program. Quantification of the protein bands was normalized by that of actin.