Fig. 6

a Immunoblot analysis of nickel-His purification of CnVA-REEP5 from transfected HEK293 cells shows stable expression of CnVA-REEP5 and successful immunoprecipitation of CnVA-REEP5 monomer (~34 kDa) and dimer (~68 kDa). b Identification of known SR/ER-shaping proteins as REEP5 interacting proteins by mass spectrometry analysis. Average precursor MS1 peak areas (peptide m/z signal) as defined by iBAQ (intensity based absolute quantification) are shown, n = 3 independent mass spectrometry runs. To calculate fold change, average null values (n.d.—not detected) were inputted with a value of 10. RTN reticulon, ATL atlastin, CKAP4 cytoskeleton-associated protein 4, ACTC1 alpha cardiac muscle actin 1, MYL6 myosin light chain 6, GAPDH glyceraldehyde-3-phosphate dehydrogenase. Asterisks indicate a statistically significant p value in a Tukey’s multiple comparison analysis where *p < 0.05. c RNASeq analysis of the RTN, ATL families of proteins, and CKAP4 in human fetal heart and adult heart tissue data using data from Human Protein Atlas. d Immunoblot analysis of nickel-His REEP5 immunoprecipitation lysates for RTN4/Nogo-A/B, ATL3, and CKAP4, n = 3. e HEK293 cells were transfected with myc-tagged Nogo-A and Nogo-B plasmids and detected with myc and α-tubulin antibodies. f Co-immunoprecipitation assay of cotransfected HEK293 cells with anti-REEP5 antibody (left panel) and anti-RTN4/Nogo-A/B antibody (right panel) demonstrated interaction between REEP5 and RTN4, n = 3. Eluates were collected on ice and loaded directly for blotting, or samples were boiled prior to blotting. g–j Co-immunoprecipitation and reverse order co-immunoprecipitation assays with g anti-REEP5, h anti-RTN4/Nogo-A/B, i anti-ATL3, and j anti-CKAP4 antibodies in adult mouse cardiac microsomes (input), followed by immunoblots analysis, n = 5. Source data containing original uncropped immunoblots are provided as a Source Data file.