Supplementary Figure 11: Investigation of the effects of JAGN1 knockdown on the protein expression levels, glycosylation and localization of neutrophil elastase (ELANE).
From: JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia
To study the effect of JAGN1 silencing on the expression of neutrophil elastase, wild-type HA-tagged ELANE was expressed in JAGN1-silenced HeLa cells. In protein blotting, neutrophil elastase and JAGN1 were detected, and actin was used as a loading control. siRNA-mediated knockdown of JAGN1 did not influence the protein expression levels or the molecular weight of neutrophil elastase (a). To study further the possible effect of JAGN1 on the glycosylation of neutrophil elastase, HA-tagged ELANE was expressed in HeLa cells in combination with siRNA-mediated JAGN1 knockdown. Treatment of protein lysates with either the EndoH or PNGase enzymes revealed no difference in the glycosylation pattern between control and siRNA-treated samples (b). Immunofluorescence studies were performed in HeLa cells to assess the potential effects of JAGN1 knockdown on neutrophil elastase (green) subcellular localization. Nuclei were visualized by DAPI staining (blue). Scale bar, 10 μm. No difference in subcellular localization between control and JAGN1 knockdown cells was detected (c).
