Modified ripa buffer

MCF-7 and MDA-MB-231 cells in LPS group were treated with 2 µg/ml LPS for 48 h. In TLR4 blocking group, 100 nmol/L eritoran was added to the cell culture for 30 min prior to the addition of 2 µg/ml LPS for 48 h. For western blot analysis, 1×10⁶ cells were lysed in modified RIPA buffer (Beyotime, Shanghai, China). The protein concentration was measured by BCA Protein Assay Kit (Beyotime) and samples were loaded on 10% SDS-PAGE. Following electrophoresis, the proteins were transferred to a BioTrace PVDF membrane (Pall Life Sciences, Ann Arbor, MI, USA) and blocked in 5% non-fat dried milk for 1 h at room temperature and incubated with rabbit antibodies against TLR4 (1∶1000; Abcam, Cambridge, MA, USA), MyD88 (1∶500, Boster, Wuhan, China) and GAPDH (1∶5000, Abcam, Cambridge, MA, USA) overnight at 4°C. After washing three times with Tris-buffered saline Tween 20 (TBST), membranes were incubated with anti-rat IgG (1∶5000, Bioworld, Dublin, OH, USA). The membrane was washed three times with TBST, and signals were detected by enhanced chemiluminescence system (Amersham Pharmacia Biotech, Bucks, UK).

Mice were anesthetized and decapitated, and brain tissue was quickly extracted. The hippocampus was rapidly dissected on ice, incubated in modified RIPA buffer (Beyotime, Nantong, China) on ice for 15 min, and centrifuged at ~200,000 × g for 10 min at 4 °C. The supernatant was collected and supplemented with 100 µl protein A/G-agarose beads overnight at 4 °C on a wheel. The beads were then allowed to sediment at the bottom of the tube, and the supernatant was collected. ZDHHC8 (1:200, Abcam), rabbit anti-GluA1 pAb (1:500; Abcam), rabbit anti-GluA2 pAb (1:200; Proteintech Group, Inc.), rabbit anti-GluA3 pAb (1:500; Bioworld Technology, Inc.), and rabbit anti-GluA4 pAb (1:200; Proteintech Group, Inc.) antibodies were incubated in equal amounts with the supernatant. Protein A/G-agarose beads were added to the supernatant, and the supernatant was incubated for 2 h at room temperature on a wheel. The beads were then washed three times with RIPA buffer and submitted to SDS-PAGE. The mixture was boiled for 10 min. The Western blots were detected using ZDHHC8 and GluA1, GluA2, GluA3, and GluA4 antibodies for immunoprecipitation^{21 (link)}.

For immunoblot analyses, modified RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) was used to extract total protein from frozen lung tissue [24 (link)]. Extraction proteins from tissues and cells were measured using the BCA protein assay reagent kit (Pierce). An equal amount of total protein (80 μg of protein/lane) was resolved on a 5–12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat dry milk in PBST (containing 0.05% Tween 20). Incubation with the antibodies was performed using the following dilutions: 1:750 for P2X₇R (Abcam, USA) and 1:1000 for caspase-1, procaspase-1, caspase-1, IL-1β (all of them from Cell Signaling Technology, USA) and total NLRP3 (Biosource, Belgium). Primary antibodies were detected with horseradish peroxidase-conjugated antibodies, 1:5000 for anti-mouse (ZSJQ-BIO, Beijing, China) and 1:5000 for anti-rabbit (ZSJQ-BIO, Beijing, China), at room temperature for 1.5 h. Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualised using a FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA). Blot bands were qualified using NIH ImageJ software.

After 48 h of transfection, cells were extracted and prepared in modified RIPA buffer (Beyotime, Shanghai, China). Total protein was extracted, and protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). A total of 20 mg of protein from each sample was used for Western blot analysis. The primary antibodies used in this study included monoclonal anti-TNFAIP3 (1:1000; Abcam, HK), anti-NF-κB/p- NF-κB (1:1000; Cell Signaling Technology, USA), anti-Bcl-2 (1:1000; Cell Signaling Technology, USA), anti-Bax (1:1000; Cell Signaling Technology, USA), and anti-capase-3/c-capase-3 (1:1000; Cell Signaling Technology, USA). β-actin was used as the loading control. Immunoreactive bands were visualized using ECL detection reagent (Millipore, Billerica, MA, USA). All data analyses were repeated thrice independently.

Total cellular proteins were extracted from the transfected cells using modified RIPA buffer (Beyotime, Shanghai, China), and protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). A total of 15 mg of protein from each sample was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with BSA in Tris-buffered saline/Tween-20, The membranes were incubated with the indicated primary antibodies: anti-PI3K/p-PI3K (1:1000; Cell Signaling Technology, USA), anti-Akt/p-Akt (1:1000; Cell Signaling Technology, USA), anti-ERK/p-ERK (1:1000; Cell Signaling Technology, USA), anti-MMP9 (1:1000; Cell Signaling Technology, USA), and anti-β-actin(1:2000;Cell Signaling Technology, USA) at 4°C overnight and then incubated with the corresponding secondary antibodies at room temperature for 2 h. Immunoreactive bands were visualized using the ECL detection reagent (Millipore, Billerica, MA, USA). All data analyses were repeated thrice independently.

To isolate the total cellular protein, the NPCs were lysed using modified RIPA buffer (Beyotime, China, Cat. No.: P0013B) which was supplemented with 1mmol/L of PMSF on ice following the manufacturer's protocol. Each protein sample (40μg) was resolved by SDS-PAGE (12%) and transferred to PVDF. After transferring, the membrane was blocked with 5% nonfat milk in Tris-buffered saline and tween 20 (TBST) at room temperature for 2h and then incubated overnight with primary anti-NLRP3(1:1000; Abcam, USA, Cat. No.: ab210491), PYCARD (1:500, Santacruz biotechnology, USA, Cat. No.: sc-514414), CASP1 (1:1000, Proteintech, Chicago, USA, Cat. No.: 22915-1-AP), IL-1β (1:500, ABclonal, Boston, USA, Cat. No.: A1112), IL-18(1:500, ABclonal, Boston, USA, Cat. No.: A1115), MAP1LC3B (1:1000, Abcam, USA, Cat. No. ab51520), SQSTM1 (1:1000, Abcam, USA, Cat. No.: ab56416), NFE2L2 (1:1000, Abcam, USA, Cat. No.: ab62352), ACTB (1:500, Santacruz biotechnology, USA, Cat. No.: sc-47778) at 4°C. The membrane was washed with TBST solution for 3 times and incubated with the secondary antibody at room temperature for 1h. The band was visualized using an ECL-Plus detection kit (New Cell and Molecular Biotech Co., Ltd, P10100). The abundance was quantified by densitometry using Quantity One software (Bio-Rad, USA).