The anti-Cul4B antibody mainly recognized the larger, unneddylated Cul4B isoforms, Cul4B-1 and Cul4B-2.A. Cul4B antibody specifically recognized a Cul4B fusion protein. Protein extracts from NPCs transfected with a construct consisting of Cul4B N-terminal 173 amino acids fused to GFP (GFP173). Left, The anti-Cul4B antibody recognized the Cul4B fragment GFP173. Right, GFP173 was confirmed by the anti-GFP blot. The * indicates the band of cleaved N-terminal GFP173.B. Analyses of Cul4B neddylation by immunoprecipitation-western using mouse brain extracts. Cul4B was immunoprecipiated with the rabbit anti-Cul4B antibody prebound to protein A-agarose. The negative control was immunoprecipitation with protein A-agarose without antibodies. Left, The blot was first probed with a rabbit anti-nedd8 antibody. A smaller band corresponding to the size of Cul4B-3 was neddylated. Right, The blot was stripped and reprobed with the Cul4B antibody. The major band corresponds to the MW of Cul4B-2. A minor band of higher MW corresponds to Cul4B-1. Cul4B-3 is also vaguely present. n = 3.C. The Cul4B antibody did not recognize Cul4A or Cul1. Cul4B was immunoprecipitated from mouse brain protein extracts as in B in triplicates. The blots were blotted with the anti-Cul4B, anti-Cul4A, or anti-Cul1 antibodies. The results show that the neddylated band in B was not Cul4A or Cul1. The results also showed that the Cul1 and Cul4A antibodies cross-reacted with Cul4B-2. The Cul4A and Cul1 bands in C are indicated by a *.D. The anti-Cul4B precipitates RBX1 in NT-2 cell extracts (n = 2) and DDB1 in mouse brain extracts. (n = 3).
Liu et al. BMC Neuroscience 2012 13:112 doi:10.1186/1471-2202-13-112