Cl xposure x ray film

The cell lysate was prepared as follows: 8 × 10⁵ fibroblasts or keratinocytes were plated in a 100 mm dish to achieve 70–80% confluence the following day. At 48 h after infection, cells were lysed with radioimmunoprecipitation assay buffer^{42 (link)}. Lysate was centrifuged at 13,500 ×g for 5 min at 4 °C, and the supernatant was mixed with a 6x Laemmli loading buffer. Before loading onto the SDS–PAGE gel, the samples were heated for 5 min at 95 °C. For C7 detection, 5–30 μg protein was loaded onto a 6% acrylamide gel. The primary antibodies used were C7 polyclonal rabbit antibody (Sigma prestige Ab, cat. no. HPA042420), GAPDH and β-actin (Santa Cruz Biotechnology). Resolved proteins were transferred onto nitrocellulose membrane with a BioRad Trans-Blot-Turbo (BioRad), blocked in PBS and 0.1% Tween with 5% milk or 5% BSA according to the requirements of the primary antibody, and incubated overnight with the primary antibody. After incubation with IgG-HRP-conjugated secondary antibody (Santa Cruz Biotechnology), the membrane was incubated with Pierce ECL western blotting substrate (ThermoFisher Scientific) and exposed to CL-XPosure X-ray film (ThermoFisher Scientific). C7 was quantified by densitometry (ImageJ v1.52), using a known concentration of purified recombinant C7 (donated by Krystal Biotech) for comparison.