Bs 0982r

Cells treated as described earlier were washed three times with PBS and lysed in RIPA buffer with a 1% protease inhibitor. Cell lysates were centrifuged, and the protein concentration was measured using a BCA assay kit. Equal protein aliquots (10 μg) were fractionated by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 3% bovine serum albumin in TBST and incubated with primary antibodies of Bax (bs-0127R, Bioss, Beijing, China), p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), PCNA (bs-2006R, Bioss, Beijing, China), LC-3 (12741S, CST, Boston, United States), Atg-5 (10181-2-AP, Proteintech, Wuhan, China), Beclin-1 (bs-1353R, Bioss, Beijing, China), NF-κB (10745-1-AP, Proteintech, Wuhan, China), p-NF-κB (bs-0982R, Bioss, Beijing, China), STING (19851-1-AP, Proteintech, Wuhan, China), cGAS (ab252416, Abcam, Cambridge, United Kingdom), iNOS (ab15323, Abcam, Cambridge, United Kingdom), GBP5 (13220-1-AP, Proteintech, Wuhan, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), and GAPDH (PMK053C, BioPM, Wuhan, China) overnight at 4°C, and then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were developed using an ECL, and the films were exposed using a bio-imaging system (170-8265, Bio-Rad).

All experiments were approved by the Administration Office of Laboratory Animals of South China Agricultural University. ICR female mice (6–8 weeks old) were housed with free food and water in a room with standard day/night cycle conditions. Piglets were purchased from Hunan New Wellful Co., Ltd. (Changsha, China). Antibodies against IL-1β (12426s), mammalian target of rapamycin (mTOR; 2972s), p-mTOR (5536s), and HIF-1α (14179s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against IκB kinase (IKK; Sc-7607), p-IKK (sc-293135), p-IκB (Sc-8404), and apoptosis-associated speck like protein containing a caspase recruitment domin (ASC; Sc-514414) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against IκB (51066-1-AP), p65 (10745-1-AP), and β-actin (60008-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). The antibody against p-p65 (bs-0982R) was purchased from Bioss (Beijing, China). Antibodies against Nod-like receptor protein 3 (NLRP3; ab214185) and caspase-1 (ab179515) were purchased from Abcam (Cambridge, UK).

Antibodies for IHC analysis included CRT (ab92516, Abcam), HSP90 (ab13495, Abcam), HMGB1 (ab79823, Abcam), PD-L1 (17952-1-AP, Proteintech), PD1 (18106-1-AP, Proteintech), CD11b (20991-1-AP, Proteintech), CD206(ab64693, Abcam), CD80 (BS-2211R,BIOSS), CD86 (ab213044, Abcam), MHCII (sc-59318, Santa Cruz), KI67 (ab16667, Abcam), PCNA (ab92552, Abcam), BAX (50599-2-lg, Proteintech), CASPASE3 (19677-1-AP,Proteintech), RAGE (bs-0177R) GBP5 (132201-AP, Proteintech), NF-κB (10745–1-AP, Proteintech), Phospho- NF-κB (bs-0982R, Bioss). Paraffin sections (5 μm) were dewaxed and rehydrated, antigen repaired with sodium citrate for 20 min, then incubated in 3% hydrogen peroxide for 10 min at room temperature. The paraffin sections were then blocked with 5% BSA for 30 min, stained with antibodies overnight at 4 °C, washed with PBS, and stained with secondary antibody (PV-9000, ZSGB-BIO) for 1 h at 37 °C. DAB (ZLI-9018, ZSGB-BIO) was applied for coloration for 5 min at room temperature. Hematoxylin was used to stain the nucleus.