Fig. 5.

(A) BiFC assay in 5-d differentiated rat primary myotubes and HeLa cells. BiFC assay was performed on 5-d differentiated myotubes (a–c) or HeLa cells (d–f) expressing either pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH1TMD (a and d) or pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH4TMD (b and e) or pBiFC-VN173-JPH4TMD and pBiFC-VC155-JPH4TMD (c and f). Scale bar, 15 µm. (B) FRAP analysis on 12-d differentiated rat primary myotubes expressing GFP-JPH1 and GFP-JPH1 deletion mutants. FRAP analysis was performed on 12-d differentiated myotubes expressing either GFP-JPH1 or GFP-JPH1 deletion mutants. Data are expressed as percentage of mobile fraction ± SEM; n values are as follows: GFP-JPH1 (n = 20); GFP-JPH1ΔMORN I-VIII (n = 12), GFP-JPH1ΔTMD (n = 7), and GFP-TMD-JPH1 (n = 10). Asterisks indicate statistical significance compared with the mobile fraction of GFP-JPH1, as evaluated by the Kruskal–Wallis multiple comparisons test (**P ≤ 0.05; ***P ≤ 0.01). (C) Immunoprecipitation experiments on HEK293T cells coexpressing myc-JPH1 and GFP-JPH1 or myc-JPH1 and GFP-JPH2, or myc-JPH2 and GFP-JPH2. Total lysates from HEK293T cells were immunoprecipitated with anti-myc conjugated agarose beads. Immunocomplexes were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by mouse monoclonal anti-myc or anti-GFP antibodies. The vertical black line at Bottom indicates that an unrelated lane was eliminated from the figure. (D) GST pull-down experiments on the microsomal fraction of mouse skeletal muscles and of HEK293T cells. A total of 500 µg of the microsomal fraction from mouse skeletal muscles or HEK293T cells expressing either GFP-JPH1 or GFP-JPH2 was used in GST pull-down experiments using GST-joining JPH1 or GST-joining JPH2 fusion proteins. Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by specific antibodies. A total of 30 µg of solubilized microsomes was loaded as control.