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. 2018 Sep 26;8:14375. doi: 10.1038/s41598-018-32685-8

Figure 1.

Figure 1

USP45 co-immunoprecipitates Spindly. (A) Endogenous USP45 was immunoprecipitated (IP) from wild-type (WT) or knock-out (KO) KBM7 cell lines. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Coomassie. The gel was divided into the indicated sections (black lines) and proteins were identified by mass spectrometry analysis. (B) Endogenous USP45 was immunoprecipitated from WT or USP45 KO KBM7 cell lines and subjected to immunoblotting with indicated antibodies. (C) Endogenous USP45 was immunoprecipitated from WT and USP45 KO U2OS cell lines and analysed as in (A). (D) Endogenous USP45 was immunoprecipitated from WT or USP45 KO U2OS cell lines and subjected to immunoblotting with indicated antibodies. (E) Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10/300 GL preparative grade column (GE Healthcare) in buffer containing 0.15 M NaCl, and every second fraction from 80 fractions obtained were denatured and analysed by immunoblotting with the indicated antibodies. (F) Fractions (5–10) of gel filtration analysis were used to immunoprecipitated endogenous USP45 and subjected to immunoblotting with the indicated antibodies. (G) Extracts of HEK293 overexpressing Spindly-FLAG were subjected to immunoprecipitation anti-FLAG and 10% of the immunoprecipitated proteins were analysed by immunoblotting for anti-FLAG and 90% for endogenous USP45. (A–G) All experiments were performed at least three times and a representative example is shown.