Cleavage of FRET-based SPs by CAPN3. (A,B) HEK293 cells were transfected with a combination of expression vectors encoding CFP, Venus, SPs (#1–#4), CAPN3WT and/or CAPN3:C129S. Immunoblot analyses revealed that intact SPs (black arrowhead in A) and cleaved SPs (white arrowhead in the blot of samples co-expressing SPs and CAPN3WT in A) were detected with an anti-GFP antibody that recognizes both CFP and Venus proteins. An anti-CAPN3 antibody captured full length CAPN3 (black arrowhead in B) and autolytic fragments of CAPN3 (white arrowhead in B). *, Non-specific bands. Note that the CFP and the Venus proteins showed slightly slow mobility compared to the SP-derived fragments since both CFP and Venus contained additional amino acid sequence before stop codon in vectors, which are absent in the SP vectors. (C) HEK293 cells were transfected with SP vectors. At 24 h post-transfection, fluorescence emission spectra from 450 to 600 nm excited by 440 nm (CFP excitation wavelength) were scanned with a fluorescence spectrometer. Emission wavelengths of the CFP (477 nm) and the Venus (528 nm) were observed in all SPs. (D) The suspension of HEK293 cells expressing SP#3 was subjected to spectral analyses. Black curve and green curve depict pre-treatment and post-treatment with ProK, respectively.

Inefficient cleavage of SP#3 by endogenous and overexpressed CAPN1 and 2. (A–C) Lysates of HEK293 cells expressing SP#3 were treated with 10 mM EDTA, 10 mM Ca²⁺ or 0.7 µM CAST at 30°C for 30 min to induce or inhibit CAPNs. Negative control, i.e. cell lysate without any treatment was shown as 0 min. Anti-CAPN1 antibody captured the full length (upper black arrowhead in A) and the autolytic fragments of CAPN1 (lower white arrowhead in A). Fodrin 150 kDa fragment was observed in Ca²⁺ treatment (white arrowhead in B). Anti-GFP antibody captured SP#3 signals of full length (black arrowhead in C) and 25 kDa fragment (white arrowhead in C). The lane labeled as CAPN3 is a positive control, i.e. SP3# co-expressed with wild type of CAPN3, which is also shown in Fig. 2A. (D–F) HEK293 cells were transfected with SP#3 expression vector in combination with either Flag-CAPN1, Flag-CAPN2, or Flag-CAPN3 (all wild type) expression vectors. Expression of Flag-CAPN1 and -CAPN2 were detected by anti-Flag antibody (gray arrowheads in D). Immunoblot by anti-CAPN1 antibody also confirmed that expression level of Flag-CAPN1 (gray arrowhead in E) was significantly higher than that of endogenous CAPN1 (a black arrowhead in E). Anti-GFP antibody captured SP#3 signals of full length (black arrowhead in F) and 25 kDa fragment (white arrowhead in F). Note that anti-Flag antibody did not detect full length of Flag-CAPN3 because of CAPN3 autolysis as shown in Fig. 2B.

Capturing activated-CAPN3 signals in ouabain treated-myotubes. (A,B) Cultured skeletal muscle cells that were isolated from wild-type (WT) mice or Capn3^C129S/C129S mice were transfected with SP#3 expression vector. At day 5–6, differentiated myotubes were treated with both 10 µM cycloheximide and 1 mM ouabain for the indicated time. Full-length bands of SP#3 (black arrowhead in A) and bands of their cleavage fragments (white arrowhead in A) were visualized by immunoblotting. Endogenous CAPN3 bands are indicated by black arrowhead (B). *, Non-specific bands. Sarcomeric α-actinin, one of the main Z-band components, was used as a loading control. CS depicts HEK cell lysate expressing CAPN3^C129S/C129S, which is a protease inactive form of CAPN3. (C) Representative image of myotubes expressing SP#3 were shown. Both images of Venus and CFP were taken under the CFP excitation wavelength. ROI were shown by white circles. Background intensity was taken from the area indicated by white rectangles. Scale bars: 20 µm. (D) The ratio of CFP/Venus in each ROI of myotubes was calculated as described in the Materials and Methods section. Black and white circles indicate the data from wild-type and Capn3^C129S/C129S myotubes, respectively. Ouabain was added to the medium at 0 min. * indicates significant difference (P<0.05) of the ratio of CFP/Venus between wild-type and Capn3^C129S/C129S. Numbers of ROI, n=69 and n=61 from 40 wild-type myotubes and 36 Capn3^C129S/C129S myotubes, respectively.