Figure 2.

Calpain 2 regulates talin proteolysis in dHL-60 cells. (A) Schematic of constructs. Stable HL-60 cell lines were generated that express FLAG-tagged versions of wild-type calpain 2 and a calpain 2 PD. Roman numerals designate the domains of calpain 2, and the active site residues within the protease domain are noted. The six EF hands in calpain 2 are shaded (dark gray). The protease-dead construct was generated by mutating the active site histidine (H262A denoted with an asterisk). (B) Expression of FLAG-tagged calpain 2 (WT) and protease-dead calpain 2 (PD) in dHL-60 cells was detected by immunoblot with α-FLAG. (C) dHL-60 cells that express wild-type calpain 1 (Capn 1), wild-type calpain 2 (Capn 2), or protease-dead calpain 2 (Capn 2 PD) were plated on coverslips coated with 2.5 μg/ml fibrinogen, stimulated with 11 nM C5a for 10 min, fixed, and stained with α-FLAG as indicated. Bar, 5 μm. (D) dHL60 cells were either not stimulated (0) or treated with C5a over a 10-min time course as indicated. Cell lysates were taken and examined for talin proteolysis by immunoblot analysis. A representative blot from three separate experiments is shown. Maximal talin cleavage was observed with 10 min of C5a stimulation. (E) Talin proteolysis was examined in dHL-60 cells that express control vector (Con), Capn 2, or Capn 2 PD. Cell lysates were obtained from dHL-60 cells that were not stimulated (NS) or treated with C5a for 10 min (C5a). A representative blot from three separate experiments is shown.