Fig. 2: ΔZFC1-SALL4 is a degradation-defective mutant.

A Schematic representation of SALL4 WT (left panel) and SALL4 mutants (right panel): ΔZFC1-SALL4 (Δ320-486 aa); ΔZFC2-SALL4 (Δ551-662 aa); ΔZFC4-SALL4 (Δ859–1028 aa); ΔNuRD-SALL4 (Δ1-12 aa). B Co-IPs of Flag-REN and ectopic SALL4 (WT or mutants) from HEK293Ts transiently transfected with indicated plasmids and C relative binding affinities (%) normalized to immunoprecipitated Flag-REN (mean of n = 3 individual experiments ± SD). D IP of HA-SALL4 from HEK293Ts transiently transfected with indicated plasmids. WT- and ΔZFC2-SALL4 are used as controls. Anti-Flag antibody was used to detect the SALL4 polyubiquitylated forms; anti-HA antibody was used to re-probe blot to assess the levels of immunoprecipitated protein. Total protein lysates are shown in the Input. E Protein levels of WT- and ΔZFC1-SALL4 in HEK293Ts transfected with a vector encoding REN and F relative densitometric analysis. G ΔZFC1-SALL4 protein stability in HEK293Ts transfected with the indicated plasmids and treated with CHX (100 µg/mL) at different time points, with H relative densitometric analysis. Representative immunoblotting of n = 3 biological replicas with similar results are shown in B, D, E, and G. Actin-normalized densitometric analysis in F and H represent the mean of n = 3 independent experiments ± SD; *p < 0.05; **p < 0.01 calculated with two-sided Student’s t-test.