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. 2012 Jul 23;287(36):30789–30799. doi: 10.1074/jbc.M112.390120

FIGURE 2.

FIGURE 2.

The polycomb protein Pc2 exerts E3 ligase activity toward hnRNP K. A, in vitro sumoylation reactions were performed using SUMO-1, -2, or -3 together with wild type or the K422R mutant T7-tagged hnRNP K. The white arrowhead denotes the migration of non-sumoylated T7-hnRNP K, while the black arrowhead indicates the migration of sumoylated T7-hnRNP K. B, HEK293T cells were transfected with wild type, K422R, or D424A mutant HA-tagged hnRNP K together with His6-tagged SUMO-2. An aliquot of cells was taken as input, and the remainder was subject to denaturing Ni2+ affinity chromatography. Both fractions were analyzed by Western blot with an anti-HA antibody. C, HEK293T cells were transfected with the indicated expression vectors and analyzed as above. D, pull-down assay was performed using HEK293T lysates expressing FLAG-Pc2. Samples were analyzed by Western blot with an anti-FLAG antibody. E, HEK293T cells were transfected with the indicated expression vectors and analyzed as above. F, HEK293T cells were transfected with His6-tagged SUMO-2 either with or without Pc2 and endogenous hnRNP K sumoylation was analyzed.