Fig. 4: Nep4 partially colocalizes with Sarcolamban A.

GFP-tagged Nep4 (mef2 > Nep4::GFP, A, A´´´´, B, C, C´´´, D, D´) and FH-tagged Sarcolamban A (mef2 > FH::SCLA, A´, A´´´´, B, C´, C´´´, D, D´) were expressed under the control of the muscle-specific mef2 enhancer and labeled with monospecific antibodies against the GFP- or the FH-tag. SERCA was labeled with a monospecific antibody detecting the endogenous protein (SERCA, A´´, A´´´´, B, C´´, C´´´, D, D´). DAPI was used as a nuclear marker (A´´´). Optical slices of third instar larval heart tissue are shown. The box in A´´´´ indicates an area of higher magnification as depicted in (B). (A–A´´´´, B) Nep4, SERCA, and SCLA signals colocalize predominantly around the nuclei (B, solid arrow). More distant from the nuclei, only SERCA and SCLA signals are visible (B, open arrows). Scale bars: 20 µm (A–A´´´´); 10 µm (B); ventral view, anterior left. (C–D´) STED images of analogously stained cardiac tissue. Boxes in C´´´ indicate areas of higher magnification as depicted in (D, D´). Overlap between Nep4, SERCA, and SCLA signals is visible predominantly around the nucleus (D, solid arrow). Individual colocalization between SCLA and SERCA (D, open arrow), Nep4 and SERCA (D, feathered arrow), or Nep4 and SCLA (D, solid winged arrowhead) occurs occasionally. Individual signals of SERCA (D, open arrowhead), Nep4 (D, open winged arrowhead), and SCLA (D, solid arrowhead) are present as well. More distant from the nucleus, only SERCA and SCLA are detected, while Nep4 is largely absent (D´). For SERCA and SCLA, again a colocalizing portion (D´, open arrow) as well as individual SERCA (D´, open arrowhead) and SCLA signals (D´, solid arrowhead) are present. Scale bars: 10 µm (C-C´´´); 1 µm (D, D´).