Fig. 4. Developmental regulation of trypanosomal clathrin and adaptin expression at the protein level. Western blot analysis demonstrates developmental regulation of TbCLH protein. (A) Antibodies generated against a fragment of TbCLH (residues 1268-1465) exhibit specific reactivity against the protein, as demonstrated by immunoprobing E. coli lysate expressing (+) or not expressing (-) a fragment of TbCLH (left). Immunoprobing of 1x10⁷ parasites with anti-TbCLH antibodies demonstrates that the heavy chain is expressed at much higher levels in BSF than PCF cells (right). Trypanosomal binding protein (BiP) is used as a loading control. (B) Reactivity of the anti-peptide antibodies to TbAPß1 was confirmed by immunoprobing yeast expressing TbAPß1. Equal lysates (2x10⁷ cell equivalents) from untransformed or transformed S. cerevisiae strain GPY418 with pGADT7ßAd were loaded per lane. The left and middle panels show yeast expressing (+) or not expressing (-) HA epitope-tagged TbAPß1 probed with anti-TbAPß1 antibody (left) or anti-HA antibody (middle). Immunoprobing trypanosomal BSF or PCF lysate with anti-TbAPß1 antibodies demonstrate approximate equivalence in protein levels (right). Scale at the left represents molecular mass in kDa (A,B).