Fig 2: INSR is required for neonatal heart regeneration.(A) Sirius red staining of P21 hearts after P1 resection, as in Figure 3. (B) Immunofluorescence detection of phosphorylated INSR at 7dpr is eliminated when Igf2 is conditionally mutated; there is no signal in the absence of injury (sham). (C) No detectable phosphorylation of IGF1R regardless of injury or Igf2 status in the 7dpr neonatal heart. Cardiomyocytes in B and C were labeled with antibodies for MYL2 (ventricular myosin light chain) or TNC (troponin C), respectively. See similar results at 3dpr for both INSR and IGF1R in Figure 4—figure supplements 1 and 2. A positive control for pIGF1R antibody quality is shown in Figure 4—figure supplement 3. (D) Western blot using ventricular lysates from P1 uninjured and resected hearts at 3dpr, comparing control to Insr conditional mutants. The phospho-specific antibody recognizes both pINSR and pIGF1R and so is labeled here simply as phospho-R. Antibodies uniquely recognizing total INSR and total IGF1R (both are approx. 95 kDa), and b-actin as a loading comparison, are also shown. The initial phospho-R blot was stripped and reprobed with the t-INSR antibody; a separate blot run at the same time using the same amount of sample was used for t-IGF1R and actin.