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Figure 3 (A) ErbB2-BirA2-HA-P2A-GFP is expressed in cardiomyocytes via the cmlc2 promoter. (B) Summary of experiment and timeline. cmlc2:erbb2-birA2-HA-P2A-GFP ventricles were collected as uninjured samples or 14 days after induced cardiomyocyte ablation (dpi). (C) Known direct interactors of ErbB2 that were captured in the ErbB2 BioID2 assay. 108 proteins showed a change >1.5-fold when normalized to uninjured cmlc2:erbb2-birA2-HA-P2A-GFP ventricles. These data were analyzed for known ErbB2 interactors. Colors and size of interactor correspond to fold changes identified during regeneration. Green: no change; blue: levels decrease; yellow: levels increase. Tns1: tensin 1 (1.57-fold); Hspb2: heat shock protein beta 2 (−2.43-fold); Gapdh: glyceraldehyde 3-phosphate dehydrogenase (p>0.05); Acta1b: actin alpha 1 (p>0.05); Ezrb: ezrin b (p>0.05); Ctnnd1: catenin δ1 (−4.59-fold); RhoA-b: Rho A-b (13-fold); Scrib: scribble (p>0.05); Anxa2a: annexin A2a (7.31-fold). (D) Over-representation test – pathway analysis of proteins increased 1.5-fold or more in ErbB2-BirA data set. p<0.001, FDR < 0.005%. Fold enrichment is shown, and protein number in red. (E) Co-immunoprecipitation of ErbB2-BirA2-HA from uninjured or regenerating cmlc2:erbb2-birA2-HA-P2A-GFP hearts. Rho A association with ErbB2 is increased after injury. Anti-HA antibody was used for ErbB2 detection, and troponin T and IgG were used as loading controls. (F) Analysis of known Rho A interactors in BirA2-GFP BioID2 data set. All known direct interactors were found to be increased when normalized to uninjured hearts. Size of circles indicates fold change; proteins are sorted clockwise after fold change from high to low. Scrib: scribble (90.9-fold); ErbB2: Erb-b2 receptor tyrosine kinase 2 (52.9-fold); Gna13: guanine nucleotide binding protein alpha 13a (25.8-fold); Rtn4a: reticulon 4a (8.82-fold); ROCK2a: Rho-associated, coiled-coil containing protein kinase 2a (5.05-fold); Ilk: integrin-linked kinase (3.91-fold); Pfn1: profilin 1 (3.25-fold); Arhgap21b: Rho GTPase activating protein 21b (2.65-fold); Wasla: WASP-like actin nucleation promoting factor a (2.5-fold); Lrp5: low-density lipoprotein receptor-related protein 5 (2.21-fold); Ctnnd1: catenin δ1 (2.2-fold); Msna: moesin a (1.9-fold). (G) Western blot analysis of ROCK2 levels in uninjured and regenerating hearts. ROCK2 levels are increased during heart regeneration.