Sc 23896

Proteins were extracted from mandibles and quantitated using a protein assay kit (Bio‐Rad, Mississauga, Ontario, Canada). Protein samples (20 μg) were fractionated by SDS‐PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described¹⁹ using antibodies against Cyclin D (1:300, sc‐8396, Santa Cruz), CDK4 (1:300, sc‐23896, Santa Cruz), p27 (1:500, sc‐1641, Santa Cruz), p53 (1:500, sc‐126, Santa Cruz) and β‐actin (1:1000, AP0714, Bioworld Technology). Bands were visualized using ECL chemiluminescence (Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corporation, NIH).

Angiotensin II (10 μM, A9525) and sauchinone (SML0783) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Four 488 phalloidin, dichlorofluorescin diacetate (DCFH-DA), DAPI, primary antibodies for p-Smad-2 (44-244G), p-Smad3 (44-246G), and NLRP3 (MA5-23919) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies for CDK2 (SC-6248), CDK4 (SC-23896), cyclinD1 (SC-8396), cyclinE (SC-247), p21 (SC-397), p27 (SC-1641), Smad-4 (SC-7966), CTGF (SC-373936), ICAM-1 (SC-8439), NfκB p65 (SC-8008), p-IκB-α (SC-8404), ASC (SC-514414), and IL-1β (SC-12742) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MCP-1 (44-246G) was purchased from Abcam (Cambridge, MA, USA). Capase-1 (4199) and HRP conjugated secondary antibodies raised against mouse, rabbit, and goat were purchased from Cell Signaling Technology (Danvers, MA, USA).

The equal amounts (500 μg) of total protein samples prepared just as described above were incubated with 30 μL Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 1 μg primary antibody, including E2F1, cdk-4 (sc-23896, Santa Cruz) or cdk-2 (sc-6248, Santa Cruz), then supplied on a rotating device overnight at 4 °C. Afterwards, the immunoprecipitates were centrifuged at 2500 rpm for 5 min at 4 °C to collect. The pellets were added 1 mL PBS to rinse three times with on a rotating device for 5 min each time. After washing finally, the pellets were re-suspended in SDS sample buffer, and subsequently boiled in 95 °C for 5 min. Furthermore, the immunoprecipitated proteins to detect antibodies against Rb, cyclin D1 (sc-426, Santa Cruz) or cyclin E (sc-481, Santa Cruz) were assayed by WB.