Figure 3

Western blot of total protein extracts of cells overexpressing DG-A170-myc^GFPand DG-K500-myc^GFP. The analysis of total protein extracts was carried out using antibodies directed against β-DG (A) and the myc-tag (B and C). A) The insertion of the myc-tag at sites A170 and K500 does not alter the expression and processing of DG, as shown by the presence of a band at ≈ 60 kDa, corresponding to β-DG-GFP (black arrow). Asterisk refers to endogenous β-DG. B) Western blot with an anti-myc antibody did not detect any signals in a total protein extract of cells transfected with K500-myc^GFP, while a band at 34 kDa (black arrow), which is likely to correspond to the α-DG N-terminal fragment harbouring the myc-tag, was present in a total protein extract of cells transfected with A170-myc^GFP. C) Immunoprecipitation with magnetic beads conjugated with an anti-myc antibody of the processed N-terminal domain of α-DG (EL) from a total protein extract of cells transfected with DG-A170-myc^GFP (TOT). The N-terminal cleavage is inhibited by CMK added to the cells (TOT + CMK).