Figure 1. CD133 can be lysine acetylated.

a) MS analysis of CD133 Flag immunoprecipitated from HEK 293/CD133-VA lysates identified CD133 to be acetylated at K216, K248 and K255. MS analysis was performed as previously described (12) with the addition that post-translational modifications for acetylation were also detected. b) Mapping of acetylated lysine sites on the predicted topology of the CD133 protein at the plasma membrane. c) Endogenous immunoprecipitation of CD133 from Caco-2 cell lysate was analyzed by Western Blot for lysine acetylation of endogenous CD133 protein. Acetylated CD133 was detected using an anti-Ac-lysine antibody (7F8, Santa Cruz Biotechnology Inc.). d) Lysates from Flag-tagged wild type CD133-F and the CD133 K-to-Q Caco-2 stable cells were Flag immunoprecipitated and analyzed by Western Blot to determine CD133 lysine acetylation. IP of CD133 was performed as previously described (12) with exception of 0.5% Triton X-100 was used for IP of CD133 for MS analysis and RIPA buffer was used for IP of CD133 for in vitro acetylation experiments in an effort to disrupt any protein-protein interactions. CD133 K-to-Q mutant was generated as previously described (3) with the following primer setsCD133(K255Q) sense 5′-CAT GGC AAC AGC GAT CCA AGA GAC CAA AGA GGC G-3′ and antisense 5′-CGC CTC TTT GGT CTC TTG GAT CGC TGT TGC CAT G-3′; CD133(K248Q) sense 5′-CCT GTT CTT GAT GAG ATT CAA TCC ATG GCA ACA GCG ATC-3′ and antisense 5′-GAT CGC TGT TGC CAT GGA TTG AAT CTC ATC AAG AAC AGG-3′; CD133(K216Q) sense 5′-CAA CAC TAC CAA GGA CCA AGC GTT CAC AGA TCT GAA C-3′ and antisense 5′-GTT CAG ATC TGT GAA CGC TTG GTC CTT GGT AGT GTT G-3′