FIGURE 3.

Calpain activation induces PTP1B cleavage which can be blocked by ALLN. Cleavage of PTP1B in HEK 293T cells can be induced by either calpain overexpression, or endogenous activation of calpain by calcium and A23187. This cleavage can be blocked by ALLN, indicating that it is calpain dependent. A, HEK 293T cells were transfected with empty vector pCMV or Flag-tagged human calpain 1 and calpain 2 (CAPN1/2) (Addgene plasmids #60941 and #60942). The cells were treated with ALLN (30 μm) for 1.5 h before harvesting. B and C, grouped densitometric data of full-length and cleaved PTP1B expression normalized to β-actin (n = 3). D, confluent HEK 293T cells were treated with ALLN for 30 min, followed by stimulation with CaCl₂ (2 mm) and A23187 (1 μm) for 1 h. E and F, grouped densitometric data after normalization to β-actin (n = 3). *, p < 0.05; **, p < 0.01. Arrows indicate full-length PTP1B protein. Asterisks indicate cleaved PTP1B under long exposure condition.