FIGURE 2.

SALL1 interacts with CBX4 in a SUMOylation-independent manner. (A) Validation of the interaction between human SALL1 and human CBX4 or Drosophila melanogaster Pc proteins using BioID-based biotin pulldown in transfected HEK 293FT cells. In the Input panel, the relative expression of the HA-tagged SALL1 proteins (the full-length protein or a TBS-related truncation mutant) is shown. One asterisk indicates SALL1-HA, while two asterisks indicate SALL1⁸²⁶-HA. Negative controls (single expression of each individual protein) are shown in lanes 4–7. Anti-GAPDH was used as loading control. As shown in the Elution panel, CBX4-BirA* interact preferentially with full-length SALL1-HA (lane 1). Anti-biotin blot shows the efficiency of the different pulldowns. (B) Validation of the interaction between SALL1 and CBX4 using GFP-Trap. The Input panel shows the expression of epitope-tagged SALL1 and CBX4 proteins in transfected HEK 293FT cells. YFP alone and HA empty vector were used as controls. Lanes 1 and 2 of the Elution panel, show that CBX4 interacts with SALL1 full length and the truncated form. (C) SUMO-related SALL1 mutants interact with CBX4. Western blot analysis of proteins extracted from HEK 293FT cells transfected with the indicated plasmids. Pulldowns were performed using GFP-Trap. As shown in the Elution panel (lanes 4, 5, and 6), interaction between CBX4 and WT SALL1 or SALL1 mutants was readily detected in all blot images. (D) Graph showing that CBX4 levels increase when co-expressed with WT SALL1-YFP, SALL1ΔSUMO-YFP, or SALL1ΔSIM-YFP. The intensity of CBX4 bands in blots was quantified using ImageJ, normalized to b-Actin and reported as fold change relative to the YFP alone control. The mean plus SEM of three independent experiments is plotted. P-values were calculated using Mann–Whitney test. *P-value < 0.05. (A–C) Antibodies used are indicated to the left. Molecular weight markers are indicated to the right in KDa.