Ab184996

Upon reaching ~60% confluence, NHEKs were treated with 2 mg/ml IgGs obtained from patients with BP (BP IgGs) or normal IgG. After 0, 0.25, 0.5, 1, 2, and 6 h incubations, the cells were washed with Dulbecco's phosphate-buffered saline (DPBS). Non-trypsin detached cells were immediately fixed with 4% paraformaldehyde and resuspended in DPBS. Cells were first incubated with a monoclonal rabbit IgG specific for human ColXVII-COOH (ColXVII IgG, ab184996, Abcam) and then incubated with anti-rabbit IgG-Alexa488 (ab150077, Abcam) to analyze the cell surface ColXVII expression. Cells were directly incubated with anti-human IgG-FITC (Dako, Copenhagen, Denmark) and examined using flow cytometry to determine the amount of IgG bound to the cell surface.

Three core genes (COL8A1, COL10A1, COL17A1), that have not been reported in NPC, were selected for western blot analysis in cell lines. Cells were collected at 90% confluence and total proteins of cells were extracted with cell lysis buffer and heated at 95 °C for 10 minutes. Then the proteins were electrophoresed and transferred to polyvinyl fluoride (PVDF) membranes (Immobilon®-P). After sealing with 5% defatted milk for 2h at room temperature (RT), the membranes were incubated with the first antibody overnight at 4℃. Then the membranes were incubated with the second antibody for 2h at RT after washing with PBST. The target bands of the membrane were visualized and photographed by smearing the chemiluminescence reagent. Then, Image J software was used to quantify the relative intensity level of the target bands. The anti-COL8A1 (ab58776), anti-COL10A1 (ab58632) and anti-COL17A1 (ab184996) were purchased from Abcam. The anti-GAPDH (10494-1-AP), HRP goat anti-rabbit IgG antibody (SA00001-2) were provided by San Ying Biotechnology, China.

hKC, JEB-KC and JEB∆6∆7 cells (1.5 × 10⁵) were seeded per well into 2-well chamber slides (Nalge Nunc International, Lab-Tek^® II Chamber Slide™ system, #154461, Rochester, NY, USA) and cultivated for 48 h in serum-free medium (CELLnTEC, #CnT-PR). At confluence, cells were fixed with ice-cold methanol for 10 min, washed with PBS and blocked with 5% BSA (Sigma, #A3294) in PBS for 1 h. The primary antibody against type XVII collagen (abcam, #ab184996) was incubated overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, #A11008, Waltham, MA, USA) and DAPI (Sigma Aldrich, #D9542) for 1.5 h at room temperature. Imaging was performed on a Zeiss LSM710 confocal microscope, and images were analyzed using ImageJ software.