Ubiquitin u 100h

The screen for UBE2s which might support histone or nucleosome ubiquitination by SHPRH was carried out using the E2Select Ubiquitin Conjugation Kit from Boston Biochem (K-982). All steps were done according to the manufacturer’s instructions; substrates (histones or polynucleosomes) were added to a final concentration of 30 ng/µl per well (in 20 µl final volume). Additional ubiquitination assays (to confirm our results and for mass spectrometry purposes) were performed in similar conditions, using UBE1, UBE2 and ubiquitin at final concentrations of 50 nM, 25 µM and 50 µM, respectively. All additional materials were purchased from Boston Biochem (Ubiquitin: U-100H; His6-Ubiquitin E1 Enzyme: E-304; Ubc5a: E2-615 and E2-616; Ubc5c: E2-625). Proteins were then separated by SDS PAGE and (ubiquitinated) histones were probed by Western Blot using the following primary antibodies: anti-H4 (New England Biolabs, 2592 S) and anti-H3 (Abcam, ab1791). IRDye secondary antibodies from LiCor (P/N 925-32210, P/N 925-68020) were used for detection using a LiCor Odyssey Imaging System.

For ubiquitination assays, an extra wash was performed using RIPA buffer supplemented with 1 M urea after immunoprecipitation. For MCL-1 ubiquitination assays performed with His-MCL-1, cells were lysed in buffer B (100 mM NaH₂PO₄, 10 mM Tris, and 8 M urea [pH 8.0]) and His-tagged MCL-1 proteins were precipitated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Valencia, CA). After washing in buffer C (100 mM NaH₂PO₄, 10 mM Tris, and 8 M urea [pH 6.3]), His-tagged proteins were eluted in buffer E (100 mM NaH₂PO₄, 10 mM Tris, and 8 M urea [pH 4.5]) and subjected to SDS-PAGE and immunoblotting. For TRAF6 in vitro ubiquitination assays, Flag-tagged TRAF6 or Tax proteins expressed in 293T cells were purified using EZview Red anti-Flag affinity gel (Sigma-Aldrich, St. Louis, MO) and incubated in ubiquitin conjugation reaction buffer supplemented with UBE1 (E-305), UbcH13 (E2-664) and ubiquitin (U-100H) purchased from Boston Biochem (Cambridge, MA) for 2 h at 30°C. For MCL-1 in vitro ubiquitination assays, Flag-TRAF6 (alone or with Tax) was purified and incubated in ubiquitin conjugation reaction buffer supplemented with UBE1, UbcH5c (E2-627), GST or GST-MCL-1, ubiquitin and energy regeneration buffer (Boston Biochem) for 2 h at 30°C. The reaction mixtures were boiled in 1× SDS sample buffer and subjected to SDS-PAGE and immunoblotting.