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. 2016 Feb 18;11(2):e0149391. doi: 10.1371/journal.pone.0149391

Fig 6. Effect of cathepsin inhibitors on the processing, activity and endocytic trafficking in T47D-MPO cells.

Fig 6

(A) T47D-MPO cells were grown with or without 8 μM ALLN for 44 h. MPO was partially purified from the resulting cell extracts on SP-sepharose and analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody to detect all MPO species (left panel) or antibodies specific for epitopes within the light chain of MPO (right panel). (B) T47D-MPO cells were grown in the presence of the indicated concentrations of e64d for 44 h and the resulting cell extracts analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody. (C) Samples analyzed in panel A were assayed for peroxidase activity and total MPO content and the relative specific activity (Active MPO/Total MPO) determined as described in “Methods and Materials”. (D) T47D-MPO cells were grown on glass coverslips in the presence of 8 μM ALLN for 72 h. The treated cells were fixed, permeabolized and double-labeled with antibodies against MPO (red) and Lamp1 (green). Mounted coverslips were imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope.