Identification of CAB063 Binding Partners

Identification of CAB063 binding partners was performed by co-immunoprecipitation. Both experimentally infected and pCI-CAB063 transfected HeLa cells were lyzed and fractionated (whole cell lysate and nuclear fraction) according to manufacturer instructions using Proteo Extract^® Subcellular Proteome Extraction Kit (Merck KGaA, Darmstadt, Germany) and Qproteome Nuclear Protein Kit (Qiagen GmbH, Hilden, Germany) as described previously (Forsbach-Birk et al., 2013 (link)). An anti PARP antibody included in the kit was used to control fractionation and identify the nuclear fraction that was used for further experiments as such (Supplementary Figure S1). 60 μg of lysate were incubated with anti CAB063 overnight, while this and further incubation steps were conducted at 4°C to reduce unspecific binding. 20 μl of sepharose beads were added thereafter and incubated for 2 h. After centrifugation and washing to remove unbound components, the sepharose-protein pellet was solubilized in Lämmli (for 1D-SDS gels) or rehydration buffer (for 2D gel electrophoresis) and heated to remove the beads prior to electrophoresis. To maximize the yield of candidate proteins, selected nuclear fractions were enriched with 2 μg of recombinant CAB063 to literally fish for eukaryotic targets.