Human APOBEC3H (A3H) haplotypes are differentially ubiquitinated. (A) Top: Immunoblot of A3H haplotypes in the absence and presence of MG132. The 293T cells were transfected with plasmids expressing A3H-HA haplotypes I through IV, as indicated. Cells were then treated with MG132 (10 µM) or equal volume of DMSO for 18 h. Western blotting of whole cell lysates was performed using anti-HA to assess for A3H levels, and anti-Lamin B1 as a loading control. Bottom: Densitometric analysis of six independent experiments. Expression of A3H-HA was normalized to Lamin B1 levels and plotted as the fold change of MG132 over DMSO control. Error bars indicate standard deviation from the mean. (B) Immunoblot of ubiquitinated A3H haplotypes. The 293T cells were co-transfected with plasmids expressing A3H-HA haplotypes, as indicated, alongside myc-tagged ubiquitin (myc-Ub). Whole cell lysates were immunoprecipitated with anti-HA resin, and Western blotting was performed with anti-HA and anti-myc antibodies. Western blotting of whole cell lysates was performed using anti-HA to confirm expression of A3H, and anti-Lamin B1 as a loading control.