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. Author manuscript; available in PMC: 2016 Jun 28.
Published in final edited form as: Cancer Lett. 2015 Mar 28;362(1):70–82. doi: 10.1016/j.canlet.2015.03.037

Fig. 2. FBXO11 promotes degradation and polyubiquitination of Snai1/2 and Scrt1.

Fig. 2

(A) FBXO11 promotes proteasomal degradation of Snai1/2 and Scrt1. HEK293 cells were transfected with Flag-tagged Snai1, Snai1-6SA, Scrt1, or untagged Snai2, together with GFP as well as empty vector, full-length or truncated FBXO11, and treated with DMSO or MG132 for 6 hours before harvest. Cellular lysates were immunoblotted with anti-Flag, anti-Snai2, or anti-GFP antibody.

(B) F-box of FBXO11 is required for degrading Snai1. HEK293 cells were transfected with Snai1, GFP, and increasing amounts of full-length FBXO11 or indicated deletion mutants. Cell extracts were subjected to immunoblotting with anti-Snai1 or GFP antibody.

(C) FBXO11 promotes polyubiquitination of Snai1 and Scrt1. HEK293 cells were transfected with Flag-tagged Snai1, Snai1-6SA or Scrt1 together with or without FBXO11, followed by treatment with MG132 for 6 hours prior to harvest. Denatured cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-ubiquitin (top) or anti-Flag (bottom) antibody. Polyubiquitinated Snai1/Scrt1 proteins were indicated as high-molecular-weight species. Immunoglobin heavy chain (from the Flag antibody used in IP) is marked by asterisks.

(D) FBXO11 accelerates Snai1 protein turnover. HEK293 cells were transfected with Snai1 and GFP, in combination with FBXO11 or FBXW1. After 24 hours, cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snai1 or anti-GFP antibody. The graph shows the quantification of Snai1 protein levels (based on the band intensity from the gels) normalized to GFP over the time course. Snai1 protein level at 0 hour time point of CHX treatment was set as 100%.