FIGURE 6.

CBX4 ubiquitination is reduced in presence of SALL1. (A) Western blot analysis of HEK 293FT cells transfected with CBX4-HA together with CMV-BirA-2A-bioUb or BirA as a negative control. Cells were treated with 50 μM of biotin in the presence or absence of 10 μM MG132. Protein lysates were subjected to pulldown with streptavidin beads and the results were analyzed by Western blot. Two asterisks indicate monoubiquitinated CBX4-HA protein and the vertical line indicates the polyubiquitination smear. (B) Western blot analysis of HEK 293FT_TripZ-SALL1-2xHA cells transiently transfected with CBX4-YFP together with BirA-2A-bioUb or BirA as control. The cells were treated or not with 1 μg/ml of doxycycline (Dox), in presence or absence of 10 μM of MG132. Protein lysates were incubated with streptavidin beads to isolate bioUb conjugated proteins and results were analyzed by Western blot. β-Actin was used as loading control. (C) The levels of ubiquitinated CBX4-YFP in Dox induced and not induced cells, in presence (right panel) or absence (left panel) of MG132, were quantified and normalized to the CBX4 levels in the input. (D) Western blot analysis of endogenous CBX4 in HEK 293FT cells transfected with CMV-SALL1-2xHA (lanes 2 and 4) or with pcDNA3 control plasmid (lanes 1 and 3), in presence (lanes 3 and 4) or absence (lanes 1 and 2) of 10 μM MG132. (E) Quantification of ubiquitinated CBX4 in the elution panel normalized to the CBX4 levels in the input, in cells expressing or not SALL1-HA, in presence (right panel) or absence (left panel) of MG132. (A,B,D) Molecular weight markers are shown to the right in KDa. Antibodies were used as indicated to the left. (C,E) Graphs represent mean plus SEM. P-values were calculated on n = 4 using Mann–Whitney test. *P-value < 0.05.