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. Author manuscript; available in PMC: 2016 Oct 31.
Published in final edited form as: Science. 2013 Jul 26;341(6144):395–399. doi: 10.1126/science.1236188

Fig. 1. SUMOylation of PTEN at lysine 254 in vivo.

Fig. 1

(A) Multiple cell lines express a 75 kDa form of PTEN. Whole cell lysates were immunoblotted with 3 PTEN antibodies: 6H2.1 mouse mAb (top), rabbit mAb CST #9559 (middle), or a rabbit pAb (bottom) CST #9552. PTEN-H is indicated (arrow). (B) Transfection of minimal PTEN cDNA leads to PTEN-H formation. pcDNA3.1-HA-PTEN (WT) or pcDNA3.1-HA (EV) were transfected into HEK293 cells and HA-immunoprecipitates immunoblotted for PTEN. (C) SUMOylation of endogenous PTEN. PTEN was immunoprecipitated from HEK293 lysates with protein A beads alone or PTEN antibody (CST #9559) and immunoblotted with the PTEN 6H2.1 antibody (left) then stripped and re-probed with antibodies to Sumo2/3 (right). (D) Formation of SUMO-PTEN requires Ubc9. HEK293 cells were transfected with scrambled or Ubc9 siRNAs, and harvested at 48 (middle lane) or 72 (right lane) hrs post-transfection. Lysates were immunoblotted for PTEN, Ubc9 and GAPDH, as indicated. Arrow indicates SUMO-PTEN. (E) Sensitivity of PTEN-H to deSUMOylases. Immunoprecipitated Flag-PTEN was incubated with GST, GST-SENP1 or GST-SENP2, as indicated, in the presence or absence of 20 mM NEM. Reactions were immunoblotted with anti-PTEN 6H2.1 (top panel) or anti-Sumo2 (lower panel) antibodies. (F) PTEN is SUMOylated on lysine 254 in vivo. HEK293 cells were transfected with FLAG-PTEN-wt or FLAG-PTEN-K254R, together with His-SUMO2 and myc-His-Ubc9, as indicated. Flag immunoprecipitates were immunoblotted for PTEN.