P p65 ser536

After treatment, protein (50 μg) from heart tissue and H9c2 cells harvested from RIPA lysis buffer were separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). A polyvinylidene fluoride (PVDF) membrane (Millipore) was used to transfer protein. After blocking with 10% non-fat milk, membranes were incubated with primary antibodies enclosing Hsp22 (3059), t-p65 (4764), p-p65^ser536 (3033), cleaved caspase-3 (9661), Bcl-2 (4223), Bax (5023), cytochrome C (4280), NLRP3 (13158), cleaved-caspase-3 (9579), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118); all were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against TLR4 (sc30002) were purchased from Santa Cruz (Dallas, TX, USA). Membranes were incubated with secondary antibodies then with enhanced chemiluminescence (ECL) reagents (170–5061, Bio-Rad). GAPDH protein was used as a reference.
TRIzol Reagent (Invitrogen) was used to isolate total RNA. cDNA was synthesized using a commercial kit (Roche). SYBR Green (Roche) was used to amplify. GAPDH RNA level was used as a reference.

MDA-MB-231 cells were grown in DMEM, and 67NR, 4T07, and 4T1 breast cancer cell lines were grown in RPMI supplemented with 10% (vol/vol) FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml; Invitrogen, Rockville, MD). The four human breast cancer cell lines MCF10A1 (M-I), MCF10AT1k.cl2 (M-II), MCF10CA1h (M-III), and MCF10CA1a.cl1 (M-IV) were obtained from Dr. Anita Roberts (NCI/NIH, Bethesda, MD). M-I, M-II, M-III, and M-IV cells were grown in DMEM/F12 (Invitrogen, Carlsbad, CA) containing 5% horse serum (Invitrogen) at 37°C with 5% CO₂. M-I and M-II cells were supplemented additionally with 10 μg/ml insulin (Sigma, St. Louis, MO), 20 ng/ml epidermal growth factor (Sigma), 0.5 μg/ml hydrocortisone (Sigma), and 100 ng/ml cholera toxin (Sigma). Antibodies specific for APP (22C11) were purchased from EMD Millipore; APP (4G8) from Covance. Specific antibodies for p27(C-19) and p21 (F-5) were from Santacruz and anti-β-actin (AC-15) was from Sigma. Antibodies purchased from Cell Signaling were AKT (#9772), pAKT Thr308 (#4056), pAKT Ser473 (#9271), pFOXO1 Thr24 (#9464), pGSK3 Ser9 (#9336), pp65 Ser536 (#3033), pERK1/2 (#9101), β-Catenin (#9562), PARP (#9542), and cleaved Caspase-3 (#9661). Anti-survivin antibody (AB8228) was purchased from Abcam. The anti-CD44 antibody (#15675-1-AP) was from Proteintech group and anti-GSK3b (KAP-ST002E) antibody was from Stressgen.

Lipopolysaccharides (LPS) from Escherichia coli was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against MAFbx, MuRF1, and α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-MyH and anti-Mstn were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies against iNOS, p38, p-p38 (Thr180/Tyr182), ERK, p-ERK (Thr202/Tyr204), JNK, p-JNK (Thr183/Tyr185), IκBα, p-IκBα (Ser32), STAT3, p-STAT3 (Tyr705), c-fos, p-c-fos (Ser32), c-jun, p-c-jun (Ser73), p65, p-p65 (Ser536), Akt, p-Akt (Ser473), p21, CDK2, cyclin D, and TBP were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3 antibody was obtained from Sigma Chemical Co., and horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies were obtained from Cell Signaling Technology.

Proteins of primary microglia or primary astrocyte were isolated using RIPA lysis buffer containing protease (HY-K0011, MedChemExpress, NJ, USA) and phosphatase inhibitor cocktail (HY-K0022, MedChemExpress, NJ, USA). After being uniformed and denatured by boiling at 95 °C for 5 min, protein samples were loaded, processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Then the membranes were blocked with 10% skimmed milk at room temperature for 1 h followed by incubating with primary antibodies against β-actin (66009-1-Ig, Proteintech, Chicago, IL, USA), PKM2, PKM1 (15821-1-AP, Proteintech, Chicago, IL, USA), C3, p-p65 ser536, p-p65 ser468 (3039, Cell Signaling Technology, Danvers, MA, USA) and p65 (6956, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After being washed with TBST, the membranes were incubated with corresponding anti-mouse (SA00001-1, Proteintech, Chicago, IL, USA) or anti-rabbit (SA00001-2, Proteintech, Chicago, IL, USA) second antibody at room temperature for 1 h and visualized by ECL chemiluminescent substrate reagent.

Cells were plated in 6 well plates at a seeding density of 1,000,000 cells/well and treated with various concentration of C800 at different time points. Cells were harvested and lysed on ice for 5 min in lysis buffer (400 mM NaCl, 0.5% triton X-100, 50 mM tris pH 7.4) and sonicated for 40 s at 10 mA. Proteins were quantified and equal amount of proteins from each group were separated in 4–15% SDS-PAGE (BioRad (Sydney, NSW, Australia)) and transferred electrically onto PVDF membrane (BioRad (Sydney, NSW, Australia)). The membrane was blocked with 5% skim milk powder in TBST, washed thrice with TBST, and incubated with specific primary antibodies overnight at 4 °C. The membranes were blotted for cyclin D1, cyclin E1, CDK4, CDK6, CDK2, p21, p-Akt (Ser473), p-Akt (Thr308), p-mTOR (Ser2448), p-p44/42 MAPK (T202/T204), and p-p65 (Ser536) (Cell Signaling Technologies, Brisbane, QLD, Australia). Dilutions were made according to manufacturer’s protocol. Then, the membrane was washed thrice with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution, GE Technologies, Brisbane, QLD, Australia) for 1 h at room temperature. The proteins were detected using Clarity Western ECL detection kit (BioRad (Sydney, NSW, Australia)). α-Tubulin (monoclonal, 1:5000, clone B512, Sigma-Aldrich, St Louis, MO, USA) was used as a loading control.

Sample preparation and SDS-PAGE were performed as previously described.^{49 (link)} The following primary antibodies were used: anti-β-actin (Sigma-Aldrich, A5441), TG2,^{50 (link)} p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-8008), p-p65^Ser536 (Cell Signaling Technology, Beverly, MA, USA, #3031) and IκBα (Cell Signaling Technology, sc-847). After the reaction with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, sc-2004 or sc-2005), immunoreactive proteins were visualized by using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). The bands were quantified using ImageJ (http://rsb.info.nih.gov/ij/).

The antibodies used and the sources were as follows: Anti-Flag M2 mouse mAb (1:5000, F1804) was obtained from Sigma (St. Louis, USA). Antibodies against TBK1 (1:1000, 3013), p-TBK1 (Ser172) (1:1000, 5483), p-p65 (Ser536) (1:1000, 3033), RIG-I (1:1000, 3743), MDA5 (1:1000, 5321), and MAVS (1:1000, 3993) were purchased from Cell Signaling Technology. Antibodies against p65 (1:2000, 10745-1-AP), β-actin (1:10,000, 66009-1-Ig), IκBα (1:2000, 10268-1-AP), and Lamin B1 (1:2000, 12987-1-AP) were purchased from Proteintech Group Inc. Lipofectamine 2000 transfection reagent and Opti-MEM were purchased from Thermo Fisher Scientific. Protease inhibitors were purchased from Roche. PMSF and DAPI were purchased from Solarbio Life Sciences, Beijing, China.