Beclin1

The lung tissues of each group were collected. Then, the protein sample was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated with primary antibody (p-Akt, Akt, p-mTOR, mTOR, Bax, Caspase-3, Caspase-9, LC-3, 1 : 1000, Cell Signaling, USA; p-GSK-3β, GSK-3β, 1 : 1000, Santa Cruz Animal Health, USA; Beclin-1, 1 : 1000, Thermo Fisher Scientific, USA) overnight at 4°C. Subsequently, the peroxidase-labeled secondary antibody (anti-rabbit IgG, 1 : 5000, Cell Signal, USA) was used for incubation for 2 h. The protein blots were visualized with an enhanced chemiluminescence (ECL) kit. Finally, the density of western blot bands was analyzed using Quantity One 1-D Analysis Software (Bio-Rad, USA).

The total protein was extracted by RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific). Next, we rinsed the beads with 100 μL iced buffer, added 100 μL antibody-binding buffer to revolve the antibody and magnetic beads for 30 min, and then rinsed the beads 3 times using 200 μL buffer for 5 min each time. Cell lysates and antibody-bound magnetic beads were incubated for 1 h at room temperature and washed using 200 μL buffer for 5 min each time. 20 μL eluent was used to rinse the beads once and the supernatant was removed. The cell lysates were extracted for Co-IP with anti-BCL2 (1:100), Beclin1 (1:100), BAX (1:100) antibodies (Thermo Fisher Scientific), and then, precipitates were detected using western blotting with anti-Beclin1 (1:1000), BAX (1:1000), BCL2 (1:1000) antibodies, respectively.

Immunohistochemical procedures were conducted according to our previous study [52 (link)]. Briefly, the deparaffinized and hydrated slides were incubated in 3 % H₂O₂ for 30 min to block the endogenous peroxidase activity and were later placed in citrate buffer (PH = 6) for antigen retrieval. The specimens were incubated with the primary antibody overnight at 4°C (p-IκBα, 1: 100; LC3B, 1: 100; Beclin-1, 1: 100, Thermo Fisher Scientific, USA; p-NF-κB p65, 1: 100; caspase-9, 1: 50; p-Akt, 1: 100; and TLR4, 1: 100; Abcam, U.K). After three washes with 0.1 mol/L PBS, the sections were incubated with a biotinylated goat anti-rabbit immunoglobulin G secondary antibody (Zhongshan Goldenbridge Biological Technology, China) at 30°C for 25 min. Next, the specimens were incubated with a streptavidin-biotin complex at 30°C for 20 min and were further incubated with diaminobenzidine for 15 min at room temperature. The slides were counterstained with hematoxylin and were visualized under a microscope at a magnification of 400× (Olympus, Germany). The positive cell counts were analyzed in five independent sections and were captured using a pathological image analyzer (Leica DM6000, Germany). The immunopositive cell rate was calculated as the number of immunopositive cells divided by the total cells (including immunopositive cells and immunonegative cells).

The following parameters were determined using the Western blotting assay: HMGB-1, AIF, LC3BII/I, and Beclin-1 (primary antibodies were purchased from Thermo Scientific Co., IL, USA), and the steps adhered to the formerly mentioned technique by El Gazar et al. (2022) [82 (link)]. Specific bands were visualized with a ChemiDocTM imaging system (Image LabTM software version 5.1, Bio-Rad Laboratories Inc., Hercules, CA, USA). The optical density (OD) of the following findings was standardized against β-actin.