Journal: Molecular Biology of the Cell
Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair
Figure Lengend Snippet: Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlin C72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlin C72 . Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl 2 . Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlin C72 is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlin C72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlin C72 , and α-40a shows reactivity to full-length dysferlin and mini-dysferlin C72 . (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.
Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).
Techniques: Generated, Polymerase Chain Reaction, Amplification, Plasmid Preparation, In Vitro, Immunoprecipitation, Incubation, Purification, Recombinant, Western Blot, Transfection, Expressing