Enhanced chemi luminescence (ecl)

Western blotting was performed as previously described, with some modifications [57 (link)]. Overnight cultures of K. pneumoniae were centrifuged and resuspended in 1 mL 50 mM Tris/HCl (pH 8.0) and ultrasonically lysed on ice (four 30-second pulses, with a 30-second pause between each pulse). The Omni-Easy Instant BCA Protein Assay Kit (EpiZyme) was used to quantify protein concentration. Proteins were electrophoresed in Tris-glycine-sodium dodecyl sulfate electrophoresis buffer (EpiZyme). Separated proteins were transferred to PVDF membranes in Omni-Flash transmembrane buffer (EpiZyme). After washing, the membranes were incubated with antibodies against AcrA (custom-made by Abmart) and GAPDH (GeneTex) overnight (GAPDH was used as an internal reference). The membranes were washed and incubated with an enzyme-linked rabbit secondary antibody (Absin). Protein bands were captured using an enhanced chemiluminescence (ECL, EpiZyme) western blotting detection system (Tanon) and densitometry was performed using Tanon Image software (Tanon).

A549 cells (5*10⁵) were seeded into 6-well plate and divided into the control group and the PPy@Fe₃O₄ group (400 μg/ml). After incubation for 24 h, the cells were washed by PBS and lysed in RIPA buffer. Then we removed the cell debris by centrifugation, and the supernatants were harvested and stored at −80°C. Equivalent amounts of protein (25 µg) were separated on 10% SDS-PAGE and transferred onto 0.22 um polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and subsequently incubated with primary antibodies including anti-MMP2/MMP9/MMP13 (Abclonal, China, 1:1,000 dilution) and anti-β-actin (Proteintech, China, 1:10,000 dilution). The following day, we washed the membranes three times for 10 min each time by Tris-buffered saline with Tween20 (TBST), and then incubated them in the corresponding secondary antibody (goat anti-rabbit IgG horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, 1:1,000, Affinity Biosciences, China) at room temperature for 2 h. Following washing three times for 10 min with TBST buffer, the membranes were treated with enhanced chemiluminescence (ECL, EpiZyme, Shanghai, China) reagent for exposure.

We added liquid nitrogen to 20–50 mg tissue, grinded it into powder, and then added an appropriate amount of protein lysis buffer (Beyotime). Whole‐cell protein lysates were extracted using protein lysis buffer, and the protein concentrations of tissue and cell were determined by the BCA assay (Solarbio). The lysates were boiled in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) sample loading buffer (EpiZyme) for 5–10 min at 99°C and separated on SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Millipore) by electroblotting, and after blocking in 5% nonfat milk (Sangon Biotech) for 2–3 h. Then the membrane was incubated with primary antibodies against anti‐MARCH1 (Immunoway), anti‐AKT (ImmunoWay), anti‐PI3K (Immunoway), anti‐pAKT (Immunoway), anti‐pPI3K (Immunoway), anti‐E‐cadherin (Cell Signaling Technology), anti‐Vimentin (Proteintech), anti‐β‐actin (Proteintech), anti‐tubulin‐α (Cell Signaling Technology) at 4°C overnight and then left with the secondary antibodies peroxidase‐conjugated goat anti‐rabbit (BOSTER) or peroxidase‐conjugated goat anti‐mouse (BOSTER) for 30 min at 37°C. Finally, the membranes were quantified using an enhanced chemiluminescence signal (ECL, EpiZyme). Photometric analyses of immunoblots were carried out using the Image Lab software package. The quantitative analysis through ImageJ software.