RNF168 ubiquitinates histone H2A but not H2B, both in vitro and in vivo. A) The in vitro ubiquitination assay was performed with GST-RNF168 wild type and the RF* mutant, using recombinant histones H2A and H2B as substrates. Refer to Methods for details. The reaction mixtures were solved by SDS-PAGE and the immunoblot with antibodies uH2A and uH2B shows the monoubiquitinated forms of histones H2A and H2B, respectively. TCL (Total Cell Lysates) are loaded to validate the signals. Immunoblots directed to RNF168 antibodies were done as control of equal loading. B) 293T cells were co-transfected with GFP alone, GFP-RNF168 WT or RF* together with FLAG-tagged histones H2A (left panels) or H2B (right panels). A small amount (1/20) of the transfected cells were lysed with standard procedure to verify protein expression level (IB using GFP and FLAG antibodies, lower panels). The remaining part was subjected to acid extraction, and the histone component was analysed by SDS-PAGE and immunodecorated as indicated. (◆) indicates the mono-ubiquitinated form of histones H2A and H2B; (◆) di- and (◆◆◆) tri-ubiquitinated forms are visible when the wild type RNF168 is expressed, but not in the presence of the RF* mutant or the vector alone. No signal of di- and tri-ubiquitination was detected for H2B. C) Cells transfected with the indicated GFP constructs were immunostained with anti-uH2A. D) 293T cells were co-transfected with cDNA encoding the GFP-RNF168 (WT) or the vector alone and the FLAG-H2A, and subjected to acid extraction. Proteins were resolved by SDS-PAGE and immunoblotted with FK2 (left panel) and Apu3.A8 (K63-chain specific, right panel) anti-Ub antibodies.