Fluorchem e

HaCat cells were cultured for 24 h and incubated with AgNPs for 24 h, and the expressions of the antioxidant protein of HO-1, and NQO1 were analyzed. Cells were lysed with RIPA lysis buffer containing 1% proteinase inhibitor to extract the total protein which was quantified by the Bradford method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the samples and transferred them to PVDF membranes. The primary antibodies (HO-1, NQO1, and β-actin) were used to incubate with the PVDF membranes at 4 °C overnight. The next day, secondary anti-mouse or anti-rabbit were then incubated for 2 h at room temperature. Proteins were visualized with the super chemiluminescence substrate ECL (Biosharp, Anhui, China) and imaged using Imager (FluorChem E, Cell Biosciences, AUS), Quantification of band intensity was performed using the Image J Software (NIH, Bethesda, MD, USA).
The procedures of the Bradford method to quantify extracted proteins. First, prepare the working solution in the desired volume ( $\mathrm{A}:\mathrm{B}:\mathrm{C}=25:24:1$ ); Second, add 145 μL of working solution and 5 μL of extracted protein lysate to 96 wells Plate, mix well, incubate at 60 °C for 20 min; Last, use a microplate reader to measure the absorbance at 562 nm; calculate the protein concentration, and use PBS to adjust the protein concentration.

Proteins of different strains were isolated using the BugBuster Master Mix according to the manufacturer’s protocol. For western blotting, protein samples in loading buffer were denatured at 100°C for 10 min and separated using SDS-PAGE. Protein bands were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked overnight in 5% nonfat milk. Anti-Flag, anti-HA, or anti-Myc antibodies (Proteintech, USA) were used as the primary antibodies in a 1:2,000 dilution with 0.5% nonfat milk in 1× Tris-buffered saline (TBS). The secondary antibody (1:5,000 dilution) was goat anti-mouse IgG (H1L)-horseradish peroxidase conjugate (Proteintech, USA). Three washes were performed in 1× TBS-Tween 20 for 10 min each, and the blots were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and photographed using FluorChem® E (Cell Biosciences, USA). To quantify the extent of phosphorylation of LuxU, blot images were analyzed with ImageJ. Intensities of both phosphorylated and unphosphorylated LuxU bands were used to calculate the fraction of LuxU∼P.

Cultured cells were collected and lysed with iced NP-40 lysis buffer (P0013F, Beyotime) containing phosphatase inhibitors (4906837001, Roche) and phenylmethylsulfonyl fluoride (PMSF) (ST506, Beyotime). The protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Immobilon‐P, IPVH00010). The membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 and then incubated with primary antibodies overnight at 4°C. The membranes were then washed and incubated with secondary antibodies for 2 h at room temperature. The primary and secondary antibodies are summarized in Supplementary Table S3. Subsequently, the membranes were scanned using an Alpha Chemiluminescence Gel Imaging System (FluorChem E, Cell Biosciences). All experiments were repeated at least three times, and the images were analyzed using ImageJ software.