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.