GFP fusion and Western blot analyses of cellular localization of CpDUF442. (A) GFP fluorescence of E. coli BL21(DE3)(pBBR2-Cpduf442-gfp) (a) and C. pinatubonensis JMP134(pBBR2-Cpduf442-gfp) (b). (B) Western blotting of CpDUF442 in cellular fractions. An anti-His tag polyclonal antibody was used as the primary antibody. Lane 1, E. coli BL21(DE3) whole-cell lysate (12 μg of protein). Lanes 2 to 4, E. coli BL21(DE3)(pET30-Cpduf442). Lane 2, soluble fraction (8.8 μg of protein); lane 3, membrane fraction (4.7 μg of protein); lane 4, whole-cell lysate (17 μg of protein).