I am investigating the levels of COL4A1 protein in a patient fibroblast cell line. Based on RNA analysis, I know the patient has a heterozygous variant resulting in aberrant RNA splicing and potential nonsense-mediated mRNA decay and thus less of the functional protein, but I would like to show that using western blotting or immunofluorescence. I have tried western blotting using multiple different COL4A1 antibodies, using cell lysates, media, and serum-free media after ascorbic acid treatment, but have not even been able to show the wildtype band in normal dermal fibroblasts. I have a positive control from mouse embryonic fibroblast media following ascorbic acid treatment that always works but none of my human dermal fibroblast samples have given me a band on western blotting. I'm wondering if anyone has had this problem or knows a better way of detecting the COL4A1 protein in human dermal fibroblasts? I'm thinking of using a loading control that is specific to the ECM but unsure which one is the best. I am also thinking of doing an immunofluorescence experiment following the treatment of the cells with protein transport inhibitors to keep the secreted COL4A1 protein and other secreted ECM proteins inside the cells to be able to detect them. Is this a good approach? Does anyone have any other recommendations? Thank you!