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. 2012 Jun;2(6):120063. doi: 10.1098/rsob.120063

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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — USP11 enhances TGFβ pathway signalling. (a) HEK293 cells stably expressing GFP or GFP-USP11 were transfected with HA empty vector or HA-SMAD7, starved for 4 h and stimulated with 50 pM TGFβ for 1 h prior to lysis. Extracts were resolved by SDS–PAGE and immunoblotted with antibodies against GFP-USP11, HA-SMAD7, endogenous phospho-SMAD2 and SMAD2. (b) HEK293 cells transiently transfected with or without HA-USP11 were starved for 4 h and stimulated with 50 pM TGFβ for 1 h prior to separation into cytoplasmic and nuclear fractions. The fractions were resolved by SDS–PAGE and immunoblotted with antibodies against HA-USP11, lamin, GAPDH, endogenous phospho-SMAD2 and SMAD2. All immunoblots are representative of at least three biological replicates. (c) TGFβ transcriptional reporter activity (using a SMAD responsive element (SRE) luciferase reporter assay) normalized to renilla-luciferase in HEK293 cells transiently transfected with SRE-luciferase, renilla-luciferase, HA-USP11, FLAG–SMAD7 and stimulated for 6 h with or without 50 pM TGFβ, as indicated. Results are average of five biological replicates. Asterisk denotes statistical significance over vector transfected and unstimulated cells. (d) TGFβ transcriptional reporter activity (using an SRE luciferase reporter assay) normalized to renilla-luciferase in HEK293 cells transiently transfected with SRE-luciferase, renilla-luciferase, HA-USP11, HA-C318S USP11 (DD), HA-USP5 and stimulated for 6 h with or without 50 pM TGFβ, as indicated. Results are average of three biological replicates. Asterisk denotes statistical significance over vector transfected and unstimulated cells. Plus symbols denote positive divergence, minus symbols denote negative divergence.