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. 2004 Mar;24(6):2499–2512. doi: 10.1128/MCB.24.6.2499-2512.2004

FIG. 5.

FIG. 5.

EGF-induced calpain activity in S50A m-calpain-expressing cells. (A) Activity of S50A m-calpain was measured in the face of down-regulation of endogenous murine m-calpain. Antisense oligonucleotides were designed to be specific for murine m-calpain. These did not block the expression of S50A m-calpain, which is human in origin. Antisense treatment in the presence of EGF for 12 h was adequate to down-regulate endogenous m-calpain (lanes 3 and 4 [from the left]), and exogenous expression of S50A was not blocked (lanes 5 to 7; n = 3). (B) Calpain activity was measured in cells expressing S50A m-calpain with the substrate Boc-LM-CMAC (5 μM). Cells that possessed the S50A plasmid but that were not induced to express it were used as a control (no dexamethasone). Control cells were not treated with murine m-calpain antisense oligonucleotides. All other cells were induced with dexamethasone (2 μM) for 18 to 24 h and treated with murine m-calpain antisense oligonucleotides for the same time period. This produced conditions under which the activity of the S50A m-calpain construct could be measured. Control cells show normal EGF-induced calpain activity (indicated by increased fluorescence, equivalent to lightness of tone in micrographs). S50A-expressing cells do not exhibit EGF-induced calpain activity, and CI-1 and the MEK inhibitor PD98059 have no additional effects (images are representative; n = 3). (C) Calpain activity was determined by a calpain activity assay kit (BioVision). WT NR6 cells expressing wild-type (WT) or S50A human m-calpain-His were pretreated for 24 h with either antisense oligonucleotides against murine (mouse AS) or human (human AS) m-calpain to down-regulate either the endogenous murine or the exogenous human m-calpain and then treated with or without CI-1 and/or EGF. The cells were lysed, and the lysates were analyzed for calpain activity. The results from this assay correlate with those from the Boc assay showing that the mutation of serine 50 to alanine significantly reduces EGF-induced m-calpain activity (P < 0.05 for endogenous mouse antisense down-regulation compared to human antisense down-regulation). Cells treated with diluent were incubated with PBS, the vehicle for EGF.