HMCN1 antibody specificity. (A) HaCaT cells were transfected with HMCN1-specific (HaCaT siHMCN1) or scramble (HaCat siControl) siRNAs. HMCN1 mRNA levels were quantified using RT-qPCR. Results were normalized to GAPDH mRNA, represent the mean ± SE of two experiments, and are expressed as a percentage of HMCN1 mRNA expression in cells transfected with siControl (two-sided t test; ***P < 0.005). (B) HaCaT cells were transfected with HMCN1-specific (siHMCN1) or scramble (siControl) siRNAs as described in A and immunostained using rabbit anti-hemicentin-1 antibody as described in Materials and methods. The negative control consisted of cells stained with the secondary antibody only. Note the decrease in hemicentin-1 staining in cells transfected with siHMCN1 (scale bar = 10 μm, hemicentin-1 [HMCN1] – red). (C) Fluorescence staining intensity in the experiment depicted in B was quantified by ImageJ. Result represents mean ± SE of two independent experiments (two-sided t test; *P < 0.05).