Ab18672

The protein expression levels were measured by western blot. In brief, total protein was obtained using the specific protein extraction kit (BestBio Institute of Biotechnology, Wuhan, China). The amounts of total protein were quantified by the BCA assay (Keygen Institute of Biotechnology, Nanjing, China). Protein was resolved by 6–15% SDS/PAGE and transferred onto poly(vinylidene difluoride) membranes (EMD Millipore, Billerica, MA, USA), then blocked with 5% nonfat milk. Primary antibodies, including P‐gp (1 : 1000; ab170904; Abcam, Cambridge, UK), STAT3 (1 : 1000; ab76315; Abcam), IL‐8 (1 : 1000; ab18672; Abcam), IL‐23 (1 : 1000; ab45420; Abcam), VEGF (1 : 1000; ab46154; Abcam), p‐STAT3 (1 : 1500; 9145; Cell Signaling Technologies, Danvers, MA, USA), MRP1 (1 : 1000; 72202; Cell Signaling Technologies), IL‐1β (1 : 1000; 12703; Cell Signaling Technologies) and p‐NF‐κB (1 : 800; sc‐136548; Santa Cruz Technology, Santa Cruz, CA, USA), were added and incubated overnight at 4 °C. The anti‐IgG secondary antibodies (ab205718, ab190475; Abcam) were subsequently applied to the membranes and incubated for 2 h. Immunoreactive signals were revealed by the enhanced chemiluminescence detection system (GE Healthcare, Muenchen, Germany). imagej software (National Institute of Health, Bethesda, MD, USA) was applied to analyze protein expressions.

Formalin-fixed paraffin-embedded blocks of tissue from patients with IgG/IgA pemphigus or conventional pemphigus were cut into 5 μm thick sections for IHC staining. These sections were stained with myeloperoxidase (MPO) (ab9535; Abcam, Cambridge, UK), C5a (ab11877; Abcam, Cambridge, UK), CD89 (ab124717; Abcam, Cambridge, UK), interleukin (IL)-8 (ab18672; Abcam, Cambridge, UK), IL-17 (ab79056; Abcam, Cambridge, UK), and matrix metalloproteinase (MMP)-9 (ab76003; Abcam, Cambridge, UK). After autoclaving for antigen retrieval, staining was performed according to the manufactures’ instructions. We used secondary antibodies conjugated to peroxidase with 3-amino-9-ethylcarbazole to detect the expression of these molecules. The sections were randomly examined and evaluated blind by a dermatologist (YTC) and a pathologist (YLC). The expression of these molecules in the epidermis was categorized from grade 0 to grade 3 based on staining intensity (Supplementary Figure S1). The number of positive infiltrating cells in the dermis was calculated by averaging the values of three randomly selected high-power fields for each specimen.

Immunohistochemical (IHC) staining was performed to detect IL-8, snail, and vimentin expression in HNSCC patient tissue samples. After deparaffinization and rehydration, the tissue slides were heated in a water bath at 100 °C with citrate buffer solution (pH 6.0) for 20 min to retrieve antigen, and then cooled at room temperature. The primary antibodies were incubated overnight at 4 °C in a humidified chamber and then visualized using a 3,3′-diaminobenzidine (DAB) detection kit (Dako Cytomation, Denmark) containing goat secondary antibody molecules and DAB chromogen. Every step of the wash used phosphate-buffered saline solution (PBS) for 5 min repeated three times. The primary antibodies (with their dilutions reported), including IL-8 (1:1000; ab18672, Abcam, USA), snail (1:1000; ab53519, Abcam, USA), and vimentin (1:200; D21H3, Cell Signaling Technology, USA) were used as the manufacturer’s instructions suggest. The intensity of the IL-8, snail, and vimentin immunoreaction was scored as follows: 0 = negative, absence of stained cells; 1 = weak; 2 = moderate; and 3 = strong. The IHC staining score was calculated by multiplying the percentage of positive cells by the staining intensity. The scoring was conducted by researchers who were blind to the clinical information of patients.

Protein extraction was performed using RIPA lysis buffer (Pierce, IL, USA) containing protease inhibitor (Roche, CA, USA). Protein extracts were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by electro-transfer to polyvinylidene difluoride membrane. After 1 h of pre-membrane blocking with 5% BSA, the proteins were incubated with respective primary antibodies at 4 °C overnight followed by secondary antibodies incubation at room temperature for 1 h. The detection of proteins was carried out using ECL reagent. Primary antibodies used in this study include: NRLP3 (1:1000, cat. no. ab214185, Abcam), METTL14 (1:1000, ab223090, Abcam), cleaved caspase-1 (1:1000, ab1872, Abcam), IL-1β (1:1000, ab2105, Abcam) and IL-18 (1:1000, ab18672, Abcam), GSDMD-N (1:1000, bs-14287R, Bioss, Beijing, China) and GAPDH (Invitrogen, cat. no. PA1-987).

TE cells were seeded onto coverslips overnight and fixed with 4% paraformaldehyde phosphate buffer solution (Wako) and incubated with rabbit monoclonal antibody against CD11b (1:100, #ab52478, Abcam, Cambridge, UK), mouse monoclonal antibody against CXCL8 (1:20, #ab18672, Abcam), rabbit polyclonal antibody against CXCR1 (1:50, #sc-988, Santa Cruz Biotechnology, Dallas, TX) and CXCR2 (1:50, #sc-682, Santa Cruz) at 4°C overnight. Alexa Fluor^® 488-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), Cy3-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories) and DyLight 488-conjugated goat anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) were incubated at room temperature for 1 h. The nuclei were stained with DAPI (Wako). Images were taken with a Zeiss LSM 700 laser-scanning microscope and analyzed using the LSM software ZEN 2009 (Carl Zeiss, Oberkochen, Germany).