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. 2008 May;56(5):495–509. doi: 10.1369/jhc.2008.950592

Figure 2.

Figure 2

Cellular processing of mCLCA6. (A) Immunoblotting analyses identified both the amino- and the carboxy-terminal cleavage products of mCLCA6 in the cell lysate (L, Lanes 1 and 5) but only the N-terminal subunit in the conditioned medium (M, Lane 2). HEK293 cells transiently transfected with mCLCA6 were analyzed by immunoblotting using antibodies αm6-N-1a (1:500) or αm6-C-1b (1:500), respectively. For analysis of the conditioned medium, a medium change was performed 42 hr after transfection followed by harvest and analysis of the medium 6 hr later. Vector alone transfected cells served as controls (mock, Lanes 7 and 8). Crosses = unspecific background staining. (B) When cells were incubated at pH 2.5, only the N-terminal cleavage product of mCLCA6 was partially released into the supernatant. Forty eight hr after transfection, HEK293 cells transiently transfected with mCLCA6 were incubated with NaCl, pH 2.5 (pH adjusted with acetic acid) to release non-covalently associated proteins from the plasma membrane. Treatment with PBS served as control. Antibodies αm6-N-1a and αm6-C-1b were diluted at 1:500. Crosses = unspecific background staining.