An interaction of heart disease-associated proteins POPDC1/2 with XIRP1 in transverse tubules and intercalated discs

Figures

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  Fig. 1. POPDC proteins pull down and co-immunoprecipitate XIRP1 and pull down annexin A5 from human myotube extract and pull-down XIRP1 from rat heart extract. Vertical black lines separate parts of blots that were originally non-adjacent or with different exposure times, as described. In all cases, the molecular weight markers in lane 1 were detected by colourimetric imaging and the antibody blots by chemiluminescence. a Glutathione beads were loaded with recombinant preparations of POPDC1, POPDC2 or not loaded (control for non-specific binding) and incubated with human myotube extracts. “Input” shows human myotube extracts diluted to 20%. Upper part of blot (A) (above 72 kDa) developed with mAb against XIRP1 (sc-166,658) shows XIRP1 in the input and pulled down by POPDC1 and POPDC2, but not in the control. The “Input” lane had a shorter exposure time than the others and the “Control” lane was originally non-adjacent. The Lower part of blot (A) (72 kDa and below) was from a separate but identical gel to the upper blot and was developed with mAb against GST tag (17A10) as a loading control. The “Control” lane was originally non-adjacent to the others. The predicted molecular weights of…

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  Fig. 2. Immunofluorescent specificity of POPDC mAbs. COS-7 cells were transfected with either (a) human POPDC1 (GFP tag) or (b) human POPDC2 (FLAG tag) and processed as described in “methods”. a mAb POPDC1 1C12 colocalised with transfected POPDC1 and (b) mAb POPDC2 12G12 colocalised with transfected POPDC2. The POPDC1 mAb did not recognise transfected POPDC2 and the POPDC2 mAb did not recognise transfected POPDC1 (not shown). c mAb POPDC1 1C12 localised to the sarcolemma of human skeletal muscle sections. This localisation was prevented by pre-incubation of the mAb with recombinant POPDC1 but not when the mAb was pre-incubated with recombinant POPDC2. Also, d localisation of mAb POPDC2 12G12 to the sarcolemma was not inhibited by pre-incubation with recombinant POPDC1 but this localisation was inhibited when the mAb was pre-incubated with recombinant POPDC2

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  Fig. 3. Immunolocalisation of POPDC1/2, XIRP1 and annexin A5 at intercalated discs and myofibrils in rat heart. In rat heart, intercalated discs were labelled with polyclonal antibody against connexin 43. a XIRP1 colocalised with connexin 43 at the intercalated disc and also stained myofibrils. b POPDC2 colocalises with XIRP1 at intercalated discs and myofibrils and POPDC2 is also present at the cardiac sarcolemma. c POPDC1 is similar to POPDC2, locating to intercalated discs and sarcolemma and to the centre of the actin I-band stained by phalloidin. d Annexin A5 colocalises with XIRP1. e POPDC2 colocalises with amphiphysin II, a marker for T-tubules

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  Fig. 4. Immunolocalisation of POPDC1, POPDC2 and XIRP1 in human heart. a POPDC1 and b POPDC2 were present at the sarcolemma and at intercalated discs. XIRP1 was also present at intercalated discs. Cross striations were difficult to resolve in the human heart sections

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  Fig. 5. Immunolocalisation of POPDC1/2, XIRP1 and annexin A5 at myofibrils in longitudinal sections of human skeletal muscle. a POPDC1 and XIRP1, b POPDC2 and XIRP1, c XIRP1 and Actinin, d Annexin A5 and XIRP1, all colocalised at myofibrils

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  Fig. 6. Immunolocalisation of POPDC1/2 and XIRP1 in transverse section of human skeletal muscle. a POPDC1 mAb 1C12 and b POPDC2 mAb 12G12 showed largely even and regular sarcolemmal staining whereas XIRP1 was often discontinuous at the sarcolemma