Alignment of SHP box sequences from human UFD1, human p47, S. cerevisiae Dfm1, and Schizosaccharomyces pombe Rbd2 C‐terminal SHP box (aa 242–251). Asterisks denote residues identical in all sequences, colons mark conservative substitutions, and dots mark semi‐conservative substitutions.
Recombinant proteins GST‐fused Rbd2 C‐terminus (Rbd2200–251), Dsc5 UBX (Dsc5323–425), Dsc2 UBA (Dsc2298–372), and GST‐HA‐V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild‐type cells. Equivalent amounts of unbound and bound fractions were probed for anti‐Cdc48, anti‐ubiquitin, and anti‐GST IgG.
GST pull‐down assay was performed as in (B) using truncated forms of Rbd2 C‐terminus; Rbd2200–251, Rbd2200–225, Rbd2225–251, and Rbd2200–240. Unbound (1×) and bound (5×) fractions were analyzed by Western blotting
Single residue mutants of conserved glycine residues in Rbd2200–251 (G244R and G246R) were tested for Cdc48 binding as in (C).
Data information: Western blots (B–D) were images using chemiluminescence.
