Fig. 2: Parkin mediates K63 ubiquitination of RIPK1.
From: Parkin regulates NF-κB by mediating site-specific ubiquitination of RIPK1
a 293 T cells were transfected with expression vectors of RIPK1 and His-Ub or His-K63 ubiquitin with or without that of Parkin for 24 h and then lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. b 293 T cells were transfected with expression vectors of His-K63 ubiquitin, RIPK1 and Parkin or K150E Parkin for 24 h and then lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. c 293 T cells were transfected with expression vectors of His-K63 ubiquitin, Flag-RIPK1 with or without that of Parkin in 293 T cells for 24 h. Flag-tagged RIPK1 was isolated by immunoprecipitation with anti-FLAG agarose beads and analyzed for ubiquitination sites by mass spectrometry. Ubiquitinated Lys residues of RIPK1 identified and quantified by mass spectrometry are listed in the Table. d 293 T cells were transfected with expression vectors of His-K63 ubiquitin, K376R mRIPK1, K550R mRIPK1, K20R mRIPK1, K105R RIPK1 with or without that of Parkin as indicated for 24 h and then lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. e 293 T cells were transfected with expression vectors of His-K63 ubiquitin and WT RIPK1 or K376R RIPK1 with or without that of Parkin for 24 h and then treated with SM164 (200 nM) or DMSO for 4 h before lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. f 0.1 mM E1 UBE1, 1 mM E2 (UbcH7), 0.1 mM ubiquitin (Boston Biochem), 5 mM MgCl2, 2 mM ATP in 50 mM Tris-HCl (pH 7.5) mixed with Flag-Parkin or Flag-K150E Parkin immunoprecipitated with anti-FLAG, and HA-RIPK1 or HA-K376R RIPK1 immunoprecipitated with anti-HA from HEK 293 cells and incubated in a total volume of 50 μL for 30 min at 37 °C
