Fig. 3

A) HOMER3 is expressed by the majority of 5637 and T24 cells (> 90%) showing mostly intracellular origin, whereas a small subpopulation also exhibits HOMER3 at the cell surface (3–5%). The left panel shows the expression of HOMER3 at the cell surface in a small subset of T24 cells (3–5%) and a massive expression of total HOMER3 (> 90% of the cells). The graph histogram at right highlights that 5637 and T24 cells presented the same HOMER3 phenotype. Experiments assessing HOMER3 at the cell surface were conducted in cells showing non-permeabilized membranes and without incorporation of propidium iodide. The analysis of permeabilized cells showed HOMER3 total levels. Results are the average of three independent replicates **** p < 0.0001 (student t-test). B) Immunofluorescence microscopy for HOMER3 (green) in non-permeabilized and permeabilized cells highlighting HOMER3 at the cell membrane (left panel). C) Immunofluorescence microscopy for HOMER3 (green) at the cell surface of non-permeabilized cells, as confirmed by a uniform expression of CellMask (orange) and the absence of signal for propidium iodine (red). Propidium iodine staining was used as cell death control for exclusion of cells with compromised plasma membranes (lower panel). In addition to PI, dead T24 cells also stained more intensely for cell mask than live cells due to dye diffusion into the intracellular space. D) Expression of HOMER3 in whole cell extracts, HOMER3 immunoprecipitates (IP) and corresponding supernatants. The blots clearly showed HOMER3 main proteoform at approximately 40 kDa, corresponding to its canonical form. HOMER3 IPs revealed additional bands at 75 and 100 kDa, potentially corresponding to glycoforms, and faint bands bellow 37 kDa. Both cell lines presented similar blot profiles. E) PNA lectin blots after in situ neuraminidase digestion (NeuAse) for whole cell extracts, HOMER3 immunoprecipitates (IP) and corresponding supernatants. Whole cell extracts show a wide number of bands spanning all molecular weights, consistent with the presence of multiple glycoproteins. Contrastingly, the IP showed bands at 75 and 100 kDa, reinforcing the existence of glycosylation. Moreover, no bands could be observed in the non-glycosylated HOMER3 canonical proteoform. Notably, several signals bellow 37 kDa were observed, suggesting low molecular weight HOMER3 glycoproteoforms. Again, both cell lines presented similar blot profiles. F) MS/MS spectra for a HOMER3 glycopeptide isolated at 75 kDa highlighting main assignments, peptide fragmentations as well as typical glycans oxonium ions at m/z 204.08 (GalNAc). G) Estimation of HOMER3 in IP bands by MS for T24 cells showed a wide number of proteoforms of distinct molecular weights (10–200 kDa), with higher abundance between 37 and 75 kDa. Bands were excised from gels, reduced, alkylated, subjected to proteolytic digestion, and analyzed by nanoLC-MS/MS. The presence of HOMER3 in each lane was estimated based on peptide-spectrum match (PSM), i.e. the total number of identified peptide spectra matched for the protein