Fig 3. Lysine lacking D_{4.2 4KR}R mutant is ubiquitinated and KLHL12 enhances its ubiquitination.

HEK293T cells were transiently transfected as indicated. 48 h post-transfection, cells were harvested and lysed. 5% of the lysate was used for IB to visualize HA D₄R, (Flag Ub)n-proteins, and Etag KLHL12, respectively (left panels). The rest of the lysates were subjected to a sequential double IP with anti-HA (16B12). Specific purification of the receptor after the second IP was confirmed upon IB with rat anti-HA (1:2000), whereas receptor ubiquitination was revealed upon IB with anti-Flag-HRP (right panels, 1:2000). The results shown are representative of four independent experiments.