Figure S4.

Reagent Validation, Cellular Localization of OTULIN, and Stable Inducible OTULIN Cell Lines, Related to Figure 4

(A) A polyclonal rabbit, anti-OTULIN antibody was generated that detects endogenous and overexpressed OTULIN in HEK 293ET cell lysates. siRNAs against OTULIN (Dharmacon pool) reduces OTULIN protein levels. Two exposures of the same western blot are shown.

(B) Comparison of the two polyclonal antisera obtained from rabbit immunization. Both antibodies detect an endogenous band that is reduced in siRNA-treated samples and that is also immunoprecipitated by both antibodies from HEK 293ET cells.

(C) OTULIN can be detected in all analyzed cell lines.

(A–C) ^*, nonspecific band.

(D) Localization of C-terminally GFP-tagged OTULIN in HEK 293ET cells. Left, DAPI stain of cell nuclei; second from left, GFP fluorescence; second from right, immunodetection of transfected OTULIN with anti-OTULIN antibody; right, merged image. Scale bar, 10 μm.

(E) GFP-tagged OTULIN was expressed in HEK 293ET cells and purified using GFP-Trap resin. Specificity assays were performed as in Figure 1D using approximately 500 nM GFP-OTULIN bound to GFP-Trap beads. A silver-stained 4%–12% SDS-PAGE gel is shown.

(F) GFP-tagged OTULIN C129A is catalytically inactive when expressed in HEK 293ET cells and purified by GFP-Trap resin.