Figure 8. Calpain 2 regulates PTP1B activity in vivo. (A) HEK cells transfected with GFP-PTP1B-HA were analyzed by Western blot after treatment with vehicle or ionomycin to stimulate calpain activity and blotted for GFP, calpain 2, talin, and actin as a loading control. Blots shown are representative of three independent experiments. The arrow indicates a calpain 2–dependent PTP1B cleavage product. (B) FLAG-PTP1B was transiently transfected into MTLn3 control and calpain 2 siRNA lines. Immunoprecipitations were performed on cells expressing FLAG-PTP1B after treatment with ionomycin. Shown are the relative amounts of FLAG-PTP1B that were pulled down. The arrow with an asterisk indicates a calpain 2–dependent PTP1B cleavage product. Blots shown are representative of three independent experiments. PTP1B cleavage was quantified for control and calpain 2 siRNA lines as a percentage of total FLAG-PTP1B expression and is shown relative to control cells. (left) Asterisks indicate statistical significance compared with control cells based on one-way ANOVA (P < 0.05). Immunoprecipitations were assayed for phosphatase activity against pNPP as described in Materials and methods. Activity of immunoprecipitated PTP1B constructs in MTLn3 calpain 2 siRNA lines is expressed relative to the control cell line. Activity data are shown as the means ± SEM from three separate experiments. (right) Asterisks indicate statistical significance based on a t test (P < 0.002). (C) MTLn3 cells were transfected with GFP-PTP1B truncated at amino acid 360 or GFP alone. Transfected cells were plated on FN-coated glass coverslips and stained with anti-cortactin antibody. Invadopodia formation was quantified by determining the mean number of invadopodia per cell. Data represent means ± SEM from three independent experiments. *, P < 0.05 compared with GFP-expressing control cells. Bar, 10 μm.
Figure 8.

Calpain 2 regulates PTP1B activity in vivo. (A) HEK cells transfected with GFP-PTP1B-HA were analyzed by Western blot after treatment with vehicle or ionomycin to stimulate calpain activity and blotted for GFP, calpain 2, talin, and actin as a loading control. Blots shown are representative of three independent experiments. The arrow indicates a calpain 2–dependent PTP1B cleavage product. (B) FLAG-PTP1B was transiently transfected into MTLn3 control and calpain 2 siRNA lines. Immunoprecipitations were performed on cells expressing FLAG-PTP1B after treatment with ionomycin. Shown are the relative amounts of FLAG-PTP1B that were pulled down. The arrow with an asterisk indicates a calpain 2–dependent PTP1B cleavage product. Blots shown are representative of three independent experiments. PTP1B cleavage was quantified for control and calpain 2 siRNA lines as a percentage of total FLAG-PTP1B expression and is shown relative to control cells. (left) Asterisks indicate statistical significance compared with control cells based on one-way ANOVA (P < 0.05). Immunoprecipitations were assayed for phosphatase activity against pNPP as described in Materials and methods. Activity of immunoprecipitated PTP1B constructs in MTLn3 calpain 2 siRNA lines is expressed relative to the control cell line. Activity data are shown as the means ± SEM from three separate experiments. (right) Asterisks indicate statistical significance based on a t test (P < 0.002). (C) MTLn3 cells were transfected with GFP-PTP1B truncated at amino acid 360 or GFP alone. Transfected cells were plated on FN-coated glass coverslips and stained with anti-cortactin antibody. Invadopodia formation was quantified by determining the mean number of invadopodia per cell. Data represent means ± SEM from three independent experiments. *, P < 0.05 compared with GFP-expressing control cells. Bar, 10 μm.

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