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. Author manuscript; available in PMC: 2014 Jun 1.

Published in final edited form as: Int J Biochem Cell Biol. 2013 Mar 1;45(6):1051–1063. doi: 10.1016/j.biocel.2013.02.015

Fig. 1 — m-Calpain cleaves ACTN4. (A) Full length WT ACTN4 expressed and purified in E.Coli were digested with indicated amount of rat m-calpain and separated by SDS-PAGE followed by either staining with Coomassie (top panel) or immunoblotting using ACTN4 antibody aa 2–11 (middle panel) and C-ter (bottom pane). (B) Graphic of circular dichroism. (C) NR6WT ACTN4 KD fibroblasts transiently expresseing FL WT ACTN4-eGFP were starved in quiescence media for 18h prior to treatment with 10 nM EGF for 15 min followed by immunoprecipitation against GFP. Immunoprecipitated protein was digested with m-calpian and then separated by SDS-PAGE followed by immunoblotting using indicated antibodies. (D) m-calpain cleavage of both FL WT ACTN4 and ACTN4 mutant Q12D. Top panel: Coomassied stained gel; Bottom panel: immunoblotting for ACTN4 using aa 2–11 antibody. The densitometry results for the FL ACTN4 immunoblotting normalized to no m-calpain are shown. (E) Both FL WT ACTN4-eGFP and ACTN4Q12D-eGFP were transiently expressed in NR6WT ACTN4 KD fibroblasts. The total cell lysate was separated by SDS-PAGE followed by immunoblotting using aa 2–11 (top panel) and C-ter antibodies (bottom panel). The densitometry results for the FL ACTN4Q12D immunoblotting normalized to FL WT ACTN4 are shown. (F) Immunoblottings of indicating protein expressed in fibroblasts. (G) Immunoblottings of FL WT ACTN4-eGFP in NR6WT ACTN4 KD fibroblasts w/wo A23187 (10uM) treatment. The densitometry results for the FL ACTN4 immunoblotting normalized to no A23187 treatment are shown. (H) Coomassie stained gel of m-calpain digested FL WT and mutant ACTN4 expressed and purified in E.coli. Images represent one of three independent experiments.

Fig. 1 — m-Calpain cleaves ACTN4. (A) Full length WT ACTN4 expressed and purified in E.Coli were digested with indicated amount of rat m-calpain and separated by SDS-PAGE followed by either staining with Coomassie (top panel) or immunoblotting using ACTN4 antibody aa 2–11 (middle panel) and C-ter (bottom pane). (B) Graphic of circular dichroism. (C) NR6WT ACTN4 KD fibroblasts transiently expresseing FL WT ACTN4-eGFP were starved in quiescence media for 18h prior to treatment with 10 nM EGF for 15 min followed by immunoprecipitation against GFP. Immunoprecipitated protein was digested with m-calpian and then separated by SDS-PAGE followed by immunoblotting using indicated antibodies. (D) m-calpain cleavage of both FL WT ACTN4 and ACTN4 mutant Q12D. Top panel: Coomassied stained gel; Bottom panel: immunoblotting for ACTN4 using aa 2–11 antibody. The densitometry results for the FL ACTN4 immunoblotting normalized to no m-calpain are shown. (E) Both FL WT ACTN4-eGFP and ACTN4Q12D-eGFP were transiently expressed in NR6WT ACTN4 KD fibroblasts. The total cell lysate was separated by SDS-PAGE followed by immunoblotting using aa 2–11 (top panel) and C-ter antibodies (bottom panel). The densitometry results for the FL ACTN4Q12D immunoblotting normalized to FL WT ACTN4 are shown. (F) Immunoblottings of indicating protein expressed in fibroblasts. (G) Immunoblottings of FL WT ACTN4-eGFP in NR6WT ACTN4 KD fibroblasts w/wo A23187 (10uM) treatment. The densitometry results for the FL ACTN4 immunoblotting normalized to no A23187 treatment are shown. (H) Coomassie stained gel of m-calpain digested FL WT and mutant ACTN4 expressed and purified in E.coli. Images represent one of three independent experiments.