Western blotting and immunoprecipitation with MAbs against hFAM76B.

(A) All MAbs except for No. 4 recognized hFAM76B-His recombinant protein expressed in E. coli. Truncated CD133-His fusion protein was used as the negative control protein and anti-His MAb was used as the positive control antibody. (B) Recombinant full-length hFAM76B from HEK 293 cells transfected with eukaryotic expressing vector carrying hFAM76B cDNA recognized by anti-FAM76B MAbs. 1and 2 indicated the samples from two independent transfection. (C) Endogenous FAM76B expressed in HepG2 or Shsy5y cells was detected by MAbs against hFAM76B; anti-GAPDH MAb was used as the positive control antibody. (D) Loss of FAM76B expression in FAM76B-/- HEK 293 cells revealed by MAbs against hFAM76B; anti-GAPDH MAb was used for loading control. (E) The cell lysates of HEK 293 cells overexpressing hFAM76B-Flag were subjected to immunoprecipitation with anti-FAM76B MAbs followed by immunoblotting with anti-Flag MAb. Anti-Flag antibody was used as a positive control and rabbit anti-mouse IgG was used as a negative control.