Hemicentin-1 directly interacts with K 14. (A) A solid growth assay was used to validate hemicentin-1–K14 interaction identified in the Y2H assay. Yeasts were transfected with pB29-HMCN1 (aa 1–212, hemicentin-1) bait vector and pP7-KRT14 (aa 92–188, K14) prey vector. Yeast colonies were grown on DO-3 media (without tryptophan, leucine, and histidine), which selects for yeast harboring interacting bait and prey proteins. Note the presence of colonies in yeasts transfected with both constructs only. (B) Flag-tagged K14 and His-tagged hemicentin-1 VWFA domain-expressing plasmids were transfected into HeLa cells. Hemicentin-1 VWFA domain and K14 were co-immunoprecipitated using anti-Flag antibody-conjugated magnetic beads followed by immunoblotting of the precipitated proteins using anti-His antibody. The experiment was repeated three times with similar results. (C) PLA was performed using antibodies directed against hemicentin-1 and K14 in healthy human skin sections. Red dots indicate positive interaction (the experiment was repeated two times). The experiment was repeated using a skin biopsy taken from individual II-2, family 4 (scale bar, 50 μm), or kidney tissue a negative control. In addition, a PLA assay using antibodies directed against keratin 5 (K5) and K14 served as a positive control (scale bars, 25 μm). Source data are available for this figure: SourceData F4.