Calpains 8 and 9 form a complex.

(A) RT-PCR analysis of calpains 8 and 9 mRNA in the WT, Capn8+/−, Capn8−/−, Capn9+/−, and Capn9−/− gastric mucosa using primer pairs: calpain 8-5′/-3′ for calpain 8, calpain 9-5′/-3′ for calpain 9, and β-actin-5′/-3′ (see Table 2). (B) Gastric mucosal homogenate was immunoprecipitated without (lane 3) or with anti-calpain 8 (lane 2), anti-calpain 9 (lane 5), or absorbed-anti-calpain 9 (lane 6) antibodies, and subjected to western blotting using anti-calpain 8, anti-calpain 9, or anti-m-calpain (for calpain 2 and CAPNS1) antibodies. Lanes 1 and 4, 2% of the input used for immunoprecipitation. (C) Elution profile of the gastric mucosal proteins of WT, Capn8−/−, and Capn9−/− mice from a Superdex 200 gel filtration column (solid line, A280). Fractions were subjected to western blotting using anti-calpain 8, anti-calpain 9, or anti-calpain 2 antibodies (lower panels). Asterisks indicate non-specific signals. (D) Gastric mucosal homogenates from WT (lanes 1–8) and Capn8−/− (lanes 9–12) mice were incubated with or without Ca2+ and inhibitors (CSTN, recombinant human calpastatin domain 1 fragment; E64, E64c). The samples were subjected to western blotting using anti-calpain 8 (lanes 1–4) or anti-calpain 9 (lanes 5–12) antibodies. Open arrowheads and asterisks indicate proteolytic fragments of calpain 9 and non-specific signals, respectively. (E) Mouse calpain 8 (lanes 1–4) was co-expressed with empty vector (lanes 1 and 3) or mouse calpain 9 (lanes 2 and 4) in COS7 cells, and mouse calpain 9 (lanes 5–8) was co-expressed with empty vector (lanes 5 and 7) or mouse calpain 8 (lanes 6 and 8). Soluble lysates (sup) were recovered by ultracentrifugation of the total lysates (total) of the treated COS7 cells. The same amounts of lysate were subjected to western blot analysis using anti-calpain 8 (lanes 1–4) or anti-calpain 9 (lanes 5–8) antibodies.