Evolutionarily conserved properties of CLCA proteins 1, 3 and 4, as revealed by phylogenetic and biochemical studies in avian homologues
Fig 4
gCLCA1 is a HExxH-dependent cleavable protein.
Immunoblot of cell lysates from HEK293 cells transiently transfected with the EYFP-mock plasmid (EYFP), the gCLCA1WT (gCLCA1) plasmid, and the gCLCA1E164Q plasmid containing an EQ substitution at position two of the catalytic active HExxH motif (gCLCA1EQ) is shown. A C-terminal cleavage product of gCLCA1 and its immature glycosylated precursor protein were identified at ~64 kDa or at ~154 kDa (*), respectively. Cleavage was prevented by the EQ substitution in the HExxH motif as no cleavage product was detectable; however, a strong band at ~166 kDa was identified, which putatively represents the uncleaved, mature glycosylated full-length protein. Identical to cells transfected with the gCLCA1WT plasmid, the immature glycosylated precursor protein at ~154 kDa was detected in cell lysate from gCLCA1E164Q transfected cells. To control for equal total protein loading the samples were identically immunoblotted with primary anti-beta-actin antibodies. Representative images of three independent experiments.