Functional Characterisation of α₁AT

<p>^Δ<b>379</b><b> Mutant.</b> (A) Immunoblot (anti-GFP) detection of α₁AT-GFP fusion protein (C-terminal tag) in whole-cell lysate and concentrated conditioned media (ie secreted) from HEK293T cells transfected with plasmids expressing either wild-type or Δ379 mutant α₁AT-GFP. Red arrow denotes position of ∼75 kDa α₁AT-GFP band, note the absence of this band in conditioned media from cells transfected with Δ379 mutant, indicating impaired secretion of mutant protein; (B) Immunoblot (anti-α₁AT) detection of α₁AT-GFP fusion protein (C-terminal tag) in whole-cell lysate, or following immunprecipitation from conditioned media (i.e. secreted) from HEK293T cells transfected with plasmids expressing either wild-type or Δ379 mutant α₁AT-GFP; (C) Transfection of either wild-type or Δ379 mutant α₁AT with an N-terminal EGFP fusion into HEK293T cells clearly indicated normal proteolytic processing of the secretion signal peptide. Both ∼75 kDA and ∼27 kDA bands are visible, representing full-length and processed (i.e. signal peptide cleaved) α₁AT-GFP fusion protein respectively; (D) At higher expression levels, accumulation of insoluble Δ379 mutant α₁AT was observed in HEK293T cells, clearly denoted by the presence of a darker band in the insoluble fraction from cells transfected with Δ379 mutant; (E) Detection of soluble (whole-cell lysate), insoluble and secreted (concentrated conditioned media) α₁AT in HeLa cells transfected with either wild-type or Δ379 mutant α₁AT-GFP. Red arrow denotes position of ∼75 kDa α₁AT-GFP band. Note the absence of this band in conditioned media from cells transfected with Δ379 mutant, indicating impaired secretion of mutant protein; (E) Fluorescent micrographs of HEK293T cells following transfection with either wild-type or Δ379 mutant α₁AT-GFP expression plasmids. Increased intracellular aggregation of mutant protein is clearly visible. NB: Loading controls represent α-tubulin immunoblot or PonceauS staining in lysate or secreted (conditioned media) samples, respectively.