Figure 1: a) Immunostaining of Ca2+ channel proteins in human skin sections (n=3, scale bar 25 µm). b) Ca2+ flux measurements upon IgG addition performed using FURA-2-AM in NHEK cells. Each curve represents 1 independent experiment, showing an average of 8 individual randomly selected measured cells, occasional non-responding cells were not included (n=4). c) Co-immunoprecipitation experiments using NHEK cells, precipitating with anti-DSG 1-IgG (n≥3). d) Co-immunoprecipitation experiments using NHEK cells, precipitating with anti-DSG 3-IgG (n≥3). e) Representative Western blot showing the phosphorylation of PLCafter 2 h IgG treatment. f) Quantification of PLC phosphorylation in Western blot after 2 h IgG treatment (n=6).
Figure 3: a) Immunostaining of DSG 1 (rabbit-pAb, Abclonal, USA) in NHEK cells. c) Immunostaining of keratin filaments in NHEK cells. White arrows: PV-IgG-induced fragmentation of DSG 1 staining or keratin and actin reorganization after incubation for 24 h; red arrows: strengthening of the cortical actin (n≥3, scale bar 25 µm).
Figure 4: a) Quantitative evaluation of blister formation in human skin slices ex vivo. b) Representative microscopic images of HE staining of human skin slices after IgG treatment for 24 h (n=3). c) Immunostaining of DSG 1 (mouse-mAb, Progen, Germany) in human skin slices after PV2 treatment. d) F-actin staining in human skin slices after PV2 treatment. Each set of images shows a 4x zoom of the blister roof and bottom. Green arrows: Missing staining at the cell border; White arrows: Remaining basal cells (tombstoning) with reduced staining (n=3, scale bar=50 μm).
Figure 5 : Schematic of the Ca2+flux-dependent signalling pathway in keratinocytes upon PV-IgG binding.