Ne pertm nuclear and cytoplasmic extraction reagents kit

Compound (+)-(R,E)-6a1 was synthesized by our group [18 (link)]. Dimethylsulfoxide (DMSO), dexamethasone (DEX), lipopolysaccharide (LPS), Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents kit was purchased from Thermo Scientific (Rockford, IL, USA).

The nuclear and cytoplasmic extraction was performed using an NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents kit (ThermoFisher Scientific, 78835, Waltham, MA, USA). A549 cells were treated with FPD5 for 24 h and were washed thrice with 1 × PBS. The cells were digested from the dish with 0.25% trypsin and collected in a 1.5 mL centrifuge tube. Cells were centrifuged at 500× g for 5 min. Then, we added 200 μL of CER1 to the cell pellet and incubated the suspension on ice for 15 min. We added 11 μL CER II, vortexed for 10 s, incubated the tube on ice for 90 s, and centrifuged at 16,000× g for 10 min. We transferred the supernatant (cytoplasm extraction) to a new centrifuge tube. We resuspended the precipitation (nucleus) with 50 μL NER after washing twice with pre-cooled PBS. We vortexed it for 20 s and incubated it for 20 min on ice, and then centrifuged it for 10 min at 4 °C, 16, 000× g. The resulting supernatant was subjected to nuclear extraction and was used for subsequent Western blot analysis.

The NE‐PERTM Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) was used to isolate and collect cytosolic and nuclear fractions following the manufacturer's protocol. Cells were lysed in radioimmunoprecipitation assay (RIPA) lysate buffer, and cell lysates were incubated on ice for 30 minutes. Cell supernatants were collected, and protein concentrations were determined using bicinchoninic acid (BCA) protein quantitation (Beyotime). SDS‐PAGE electrophoresis was performed on proteins from cell lysate proteins and transferred to PVDF membranes. Membranes were incubated with primary antibody overnight at 4℃. The primary antibodies and secondary antibodies are shown in Table S2. Proteins in membranes were visualized using an enhanced chemiluminescence kit (BOSTER).
For IHC, mouse tumour sections were dewaxed and rehydrated. The antigen was retrieved under high pressure using citrate buffer (pH = 6.0). The Ultra‐sensitive S‐P kit (Maixin‐Bio) was used to block endogenous peroxidase activity and reduce non‐specific reactivity. Sections were then incubated with primary antibodies (shown in Table S2) at 4°C overnight. Mouse tumour sections were then incubated with secondary antibody and streptomycin avidin‐peroxidase using the Ultra‐sensitive S‐P kit, and the sections were visualized with DAB reagent (Maixin‐Bio).