Fig 2: Co-immunofluorescence analysis demonstrates colocalization of FAM162A and COX20 with known mitochondrial marker, COXIV, and MCT1 colocalization with known plasma membrane protein, Gai, in isolated adult mouse cardiomyocytes. (a) Immunofluorescence analysis of FAM162A (green) co-stained with mitochondrial protein, COXIV (red) in acutely isolated adult mouse cardiomyocytes. Three-dimensional reconstructive analysis demonstrates regions of colocalization (yellow) with a Pearson’s coefficient p > 0.5. Scale, 10 µm. (b) Immunofluorescence analysis of MCT1 (green) co-stained with known plasma membrane protein, Gai (red) in acutely isolated adult mouse cardiomyocytes. Three-dimensional reconstructive analysis demonstrates regions of colocalization (yellow) with a Pearson’s coefficient p > 0.5. Scale, 10µm. (c) Immunofluorescence analysis of COX20 (green) co-stained with mitochondrial protein, COXIV (red) in acutely isolated adult mouse cardiomyocytes. Three-dimensional reconstructive analysis demonstrates regions of colocalization (yellow) with a Pearson’s coefficient p > 0.5. Scale, 10µm. Nuclear staining was visualized with Hoechst staining (blue). All images shown are representative of approximately 30–40 total images captured per condition, n = 3 independent biological replicates. All original uncropped microscopy images were uploaded to figshare (10.6084/m9.figshare.11844972.v12).

Fig 3: Immunblot analysis of protein expression across multiple tissues and microsomal fractions. Immunoblot analysis of (a) FAM162A, (b) MCT1, and (c) COX20 protein expression levels in mouse tissues. Blots were incubated with either anti-DDK antibody or FAM162A, MCT1 or COX20. Immunodepletion experiments were carried out by incubating primary antibodies together with HEK overexpression lysates. Coomassie blue gels were performed to visualize protein loading across all samples. (d) Immunoblot analysis of FAM162A, MCT1, and COX20 in isolated adult cardiac cytosolic fraction and enriched membrane microsomes from 10 mouse hearts. Blots were probed with primary antibodies as indicated, and in parallel, samples were run on a Coomassie blue gel. All immunoblots shown are representative of 3 total immunoblots performed, n = 3 independent biological replicates. Original uncropped immunoblots are provided in Supplemental Fig. 9. All original uncropped images were uploaded to figshare (10.6084/m9.figshare.11844972.v12).

Fig 4: Membrane topology and prediction analysis of FAM162A, MCT1, and COX20. (a–c) Prediction of human FAM162A, MCT1, and COX20 protein topography generated by TOPCONS (http://topcons.cbr.su.se). (d–f) Predicted membrane topology model of human FAM162A, MCT1, and COX20A generated by modification of a T(E)Xtopo output. Peptide immunogen sequences for commercially available FAM162A, MCT1, and COX20 antibodies used in the current study are indicated by an antibody icon.

Fig 5: Immunohistochemistry analysis of top ranked cardiac-enriched candidates. Immunohistochemistry analysis of the top ranked candidates in each subcellular classification (Mitochondrion, plasma membrane, other organelles (ER, golgi apparatus, peroxisomes, lysosomes), cytosol, and the secretory pathway in healthy human ventricular tissues from the Human Protein Atlas. Immunohistochemical stain intensity was assessed independently as either weak, moderate or strong and reported as protein expression score (PES). COX20, moderate PES; FAM162A, weak PES; Tmem65, moderate PES; MCT1, moderate PES; SLC12A7, moderate PES; TLN2, moderate PES; MSN, moderate PES; FLOT1, moderate PES; SNTA1, moderate PES; DYSF, moderate PES; AOC3, negative PES; REEP5, moderate PES; STOM, weak PES; CTNND1, moderate PES; LAMA2, strong PES; LAMB2, strong PES; BGN, moderate PES; MYH7, strong PES; TNNT2, strong PES; TNNI3, strong PES. Scale bar, 25μm. All images shown are representative of approximately 5 distinct regions of interest (ROIs) assessed per sample, n = 3 independent patient tissue blocks.