In Vivo Ubiquitination Assay

In vivo ubiquitination assay was performed as described in our previous publication (29). Briefly, HEK-293T cells were seeded in 90 mm culture dishes to reach for almost 70% confluency before transfection. 5µg of pLV-IRF9 plasmids and 5µg of 6X-His-ubiquitin plasmids were transfected along with 5µg of Flag-HA-USP33 in co-transfection experiments to check the impact of USP33 on ubiquitination levels of IRF9. After 24 hrs of transfection, all cells were treated with 20µM final concentration of MG132 and incubated for at least 8 hours. Cells were then harvested and sonicated and lysed with Buffer A (6M guanidine-HCL, 0.1M Na2HPO4, 10mM imidazole at pH 8.0). The lysates were then incubated with Ni-NTA agarose beads overnight on rotor shaker at room temperature. The beads were washed next morning with buffer A, followed by Buffer A+Ti (1 volume of buffer A and 3 volume of buffer Ti (25mM Tris-HCL, 20mM imidazole at pH 6.8) and finally with buffer Ti. Final elutions were done with 50µl of His-Ubiquitin elution buffer (0.2M imidazole, 5% w/v SDS, 0.15M Tris-Cl at pH 6.8) also known as 2X Laemmli buffer and boiled for 5 minute at 100°C. Samples were run on SDS gel and probed with anti-IRF9 antibody.