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. 2017 May 3;15(5):e2001220. doi: 10.1371/journal.pbio.2001220

Fig 4. Suppressor of Mek null (Smek) promotes methyl-CpG–binding domain protein 3 (Mbd3) degradation and polyubiquitylation.

Fig 4

(A) HEK293 cells and cell lines stably expressing Smek1 or Smek2 were treated with cycloheximide (CHX) for indicated times and whole cell lysates were prepared for immunoblotting (n = 4). (Lower) Quantification of band intensities is shown in the lower panel. (B) Wild-type and Smek1/2 dKO NPCs were treated with CHX for indicated times (h, hours) and harvested, and then lysates were immunoblotted (n = 3). (Lower) Quantification of band intensities. OE, overexpression; Ub, ubiquitin. (C) Ubiquitylation assay of Mbd3 using control and Smek1 or Smek2 stable HEK293T transfected with Mbd3-myc and HA-Ub expression vectors and treated 1 d later with MG132 for 6 h (upper blot) and IB for indicated proteins (lower blot). (Lower) Quantification of band intensities (n = 3). (D) Same as panel C except using WT or Smek1/2 dKO NPCs (n = 2). (E) Same as panel D except using cells transfected with Mbd3(ΔN92)-myc and HA-Ub expression vectors at control and Smek1 or Smek2 stable cell lines (n = 2). In (A–C and E), quantification of band intensity was done using ImageJ. Data are presented as average ± SD. Statistical t test analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.005; not significant (ns), p > 0.05). All quantification data underlying panels A, B, C, and E can be found in S2 Data.