Ab6759

Total proteins in human cSCC cell lines (A431 and SCL-1 cells) and human normal skin HaCaT cells were extracted by RIPA lysate, and the concentrations of the proteins were calculated by BCA protein concentration determination kit. Protein samples were separated by electrophoresis before transferring to PVDF membrane. The membrane was incubated in 5% skim milk at indoor temperature for 2 h, followed by incubation with primary antibodies at 4°C overnight. Then, the membrane was incubated with HRP-coupled secondary antibody (ab6759, Abcam) for 2 h at room temperature. ECL was developed and exposed in gel imaging system. The gray value of band was measured by Image-Pro Plus 6.0 software with GAPDH as reference. The relative expression level of the target band was obtained by the gray ratio with the internal reference.

Immunocytochemistry was performed with KYSE-30 and YM-1 cells expressing TWIST1 or GFP. The cultured cells were trypsinized and the cell suspension (10⁶ cells per slide) was pelleted onto poly-L-lysine-coated glass coverslips using a cytospin centrifuge (Thermo Fisher Scientific). The coagulated cell mass was fixed using 4% paraformaldehyde (PFA) for 15 min. Cells were permeabilized with 0.01% Triton-X-100 in PBS for 10 min, followed by blocking with 1% bovine serum albumin (BSA) in PBS for 45 min at RT. Subsequently, cells were incubated with the following primary antibodies overnight at 4 °C: Anti-E-Cadherin (Abcam, ab15148, 1:1000 dilution), Anti-vimentin (Abcam, ab137321, 1:1000 dilution), Anti-β-catenin (Abcam, ab2365, 1:1000 dilution), Anti-Bcl2 (Abcam, ab59348, 1:1000 dilution), Anti-Bax (Abcam, ab53154, 1:1000 dilution). After washing, cells were incubated with HRP-conjugated rabbit anti-human IgG (Abcam, ab6759, 1:2000 dilution) for 30 min followed by incubation with the chromogen 3,3′-Diaminobenzidine (DAB) for 10 min at RT. Counterstaining was performed using Mayer's hematoxylin.

ELISA was conducted to determine the antibodies and titres of serum binding antibodies to SARSCoV-2 RBD. Corning 96-well Stripwell Flat Bottom Microplates (Corning® 9102) were coated with 2.5 μg/mL SARS-CoV-2 RBD protein overnight at 4 °C. Plates were washed 5 times the next day with PBST (PBS containing 0.05% Tween-20) to remove unbound RBD protein and then blocked with 5% skim milk (Biotopped, D6340) in PBST for 2 h at 37 °C. For tilter, twofold serially diluted plasma were added to the wells and incubated for 1 h at 37 °C. For RBD specific antibodies, plasma was added to the wells after 1:500 dilution in 5% milk and incubated for 1 h at 37 °C. For IgG, Rb pAb to Hu IgG (HRP) antibody (abcam, ab6759) was used at a 1:10,000 dilution. For IgM, Rb pAb to Hu IgM (HRP) antibody (abcam, ab97210) was used at a 1:8000 dilution. For IgA, Rb pAb to Hu IgA (HRP) antibody (abcam, ab73901) was used at a 1:2000 dilution. For IgG subsets, Mouse anti-human IgG1-4 Fc secondary antibody (nitrogen, MH1715, MH1722, MH1732, MH1742) was used at a 1:200 dilution. Plates were washed 5 times with PBST. Plates were developed with TMB Two-component Substrate solution (solarbio, PR1210) for 5–30 min at room temperature. The reaction was stopped with ELISA stop solution. Plates were read on a Spectramax Plate Reader at 450 nm using Thermo Scientific Multiskan SkyHigh.

Proteins that separated from RA‐FLSs with RIPA lysis buffer (Shanghai Absin Biotechnology Co., Ltd.) were quantified with the application of a bicinchoninic acid (BCA) protein assay kit (Shanghai Yisheng Biotechnology Co., Ltd.). Then, the proteins were exposed to 8% SDS‐PAGE, after which were transferred to PVDF membranes. The membranes that sealed by 5% nonfat milk or 5% bovine serum albumin (BSA) were subjected to primary antibodies against SPTBN1 (ab124888; 1:1,000; Abcam), MMP2 (ab92536; 1:1,000; Abcam), MMP9 (ab76003; 1:1,000; Abcam), Bcl2 (ab32124; 1:1,000; Abcam), Bax (ab32503; 1:1,000; Abcam), cleaved caspase3 (ab32042; 1:500; Abcam), PIK3R2 (ab180967; 1:2,000; Abcam), p‐PI3K (ab278545; 1:1,000; Abcam), p‐AKT (ab38449; 1:1,000; Abcam), PI3K (ab140307; 1:1,000; Abcam), AKT (ab8805; 1:1,000; Abcam), or GAPDH (ab9485; 1:2500; Abcam) at 4°C overnight, after which was the probe with HRP‐labeled goat anti‐rabbit secondary antibody (ab6759; 1:5000; Abcam) at room temperature for 2 h. At last, ECL (Yeasen Biotech) and ImageJ (Version 146) were applied for the visualization and analysis of protein blots.