β actin

H413 clone-1 cells were treated with different conditions as described above. Whole cell proteins were prepared by scraping the cells in cold PBS, extracted in SDS sample buffer, separated by SDS-PAGE using 5% to 12% gradient mini-gels, transferred to nitrocellulose membranes (Bio-Rad), and blocked overnight with 3% BSA (Sigma) in 0.1 mol·L^-1 Tris buffered salts solution pH 7.4 (TBS). The nitrocellulose membranes were then incubated with rabbit polyclonal antibodies to human occludin (ab31721), JAM-A (ab106114), claudin-1 (ab15098), claudin-4 (ab53156), ZO-1 (ab59720), claudin-15 (ab215354; all 1 μg·mL⁻¹, Abcam, Cambridge, UK), and β-actin (0.1 μg·mL⁻¹, GenTex, Zeeland, MI, USA) in 0.05% Tween20/TBS for 4 h. β-actin was used as a loading control. After the incubation, the membranes were washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1 500 in Tween20/TBS for 2 h. Bound antibodies were displayed with AP substrate (Bio-Rad) after the development of reactivity for proteins.

The proteins of tissue and cells were extracted using Protein Extraction Reagent (78510; ThermoFisher), and quantified using BCA Protein Assay Reagent (Beyotime). The primary antibodies as follows: β‐actin (GTX109639; GenTex), CB2 (ab3561; Abcam), Cyclin A2 (ab181591; Abcam), Bcl2 (D17C4; Cell Signaling Technology [CST]), Bax (D2E11; CST), phosphorylated (p)‐Akt (D9E; CST), Akt (11E7; CST), p‐mTOR (D9C2; CST), and mTOR (7C10; CST) overnight at 4°C, then, goat anti‐rabbit secondary antibodies (A00834; Multi Sciences) was incubated for 1 h. Finally, images were exposed using an enhanced chemiluminescence kit (Chemstudio SA2, Analytik Jena).

To measure inflammasome protein expression, 2- and 4-h cultures of the different conditions (treated with P. gingivalis, P. gingivalis LPS, ATP 3 h + P. gingivalis or ATP 3 h + P. gingivalis LPS) were extracted in SDS sample buffer and separated by PAGE using gradient 5 to 12 % mini-gels, transferred to nitrocellulose membranes (Bio-Rad) and blocked overnight with 3 % BSA (Sigma) in 0.1 M Tris buffered salts solution pH 7.4 (TBS). Blotted antigens were incubated with rabbit polyclonal anti-human antibodies CIAS1/NALP3, TMS1/ASC, Caspase-1 (1 μg/ml, Abcam, Cambridge, UK), and β-actin (0.1 μg/ml, GenTex, Zeeland, MI, USA) as a loading control in 0.05 % Tween20/TBS for 4 h, washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1500 in Tween20/TBS for 2 h. Bound antibody was visualized with AP substrate (Bio-Rad) after development of reactivity for proteins from control antibody.