Anti nf κb p65 sc 8008

LPS (L2630, E. coli 0111: B4) was purchased from Sigma-Aldrich (St. Louis, MO, United States). MaR1 was obtained from Cayman Chemical (Ann Arbor, MI, United States). The primary antibodies used in this study were as follows: anti-NOX4 (ab133303, Abcam), anti-TNF-α (AF7014; Affinity), anti-IL-6 (347023; Zenbio), anti-MCP-1 (AF-479-SP, R&D), anti-BAX (50599-2-Ig; Proteintech), anti-BCL-2 (26593-1-AP; Proteintech), anti-cleaved caspase-3 (9661; Cell Signaling Technology), anti-DRP-1 (NB110-55288; Novus), anti-MFN-1 (13798-1-AP; Proteintech), anti-OPA-1 (NB110-55290; Novus), anti-p-IκBα (AF2002; Affinity), anti-IκBα (AF5002; Affinity), anti-p-NF-κB P65 (3033; Cell Signaling Technology), anti-NF-κB P65 (sc-8008; Santa Cruz), and anti-GAPDH (ab8245, Abcam).

Briefly, cells were lysed by RIPA (Beyotime, Shanghai, China) and the cell lysate was added with antibodies. After incubation at 4 °C overnight, Protein A/G PLUS Agarose (Santa Cruz Biotechnology, USA) was added into the mixture and incubated at 4 °C overnight. The proteins were eluted and analyzed by western blotting. Antibodies used for co-IP were listed as follows: anti-DHX15 (sc-271686, Santa Cruz Biotechnology, USA) and anti-NF-κB p65 (sc-8008, Santa Cruz Biotechnology, USA).

The antibodies against PD-L1 (#13684S), CD276 (#14058S), CD47 (#63000S), p-STAT3 (Y705) (#9145S), p-STAT1 (Y701) (#9167S), p-STAT1 (S727) (#8826S) and Galectin-9 (#54330S) were purchased from Cell Signaling Technology (Beverly, ME, United States). Antibodies against STAT1 (ET1606-39), p-NF-κB p65 (S529) (ET1604-27), p-c-Myc (S62) (ET1609-64), p-c-Myc (T58) (ET1611-24), c-Myc (RT1149) were purchased from HuaBio (Hangzhou, China). Anti-STAT3 (10253-2-AP) was purchased from Proteintech (Proteintech, Chicago, IL, United States). Anti-NF-κB p65 (sc-8008) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH (db106) was purchased from diagbio (Hangzhou, China). Bafetinib (T6311), MG132 (T2154), Chloroquine (T8689), and CMC-Na (T19232) were purchased from TargetMol (Boston, MA, United States). FITC-conjugated CD274 mouse monoclonal antibody (#558065) and FITC mouse IgG were purchased from BD Biosciences (San Jose, California, United States).

RAW264.7 cells at the density of 1 × 10⁵ cells/well were seeded in a 12‐well plate and were pretreated with 10 μM of eriodictyol for 24 hr, followed by incubation with LPS (1 µg/ml) and PMA (100 ng/ml) for 30 min. After treatments, the cells were fixed with 4% paraformaldehyde solution for 30 min, permeabilized in 0.1% Triton X‐100 for 5 min, and blocked with 5% BSA for 1 hr at room temperature. The fixed cells were further incubated with anti‐NF‐κB (p65) (sc‐8008, Santa Cruz Biotechnology) antibody for 2 hr, followed by washing with PBST 3 times. The specimens were then stained 1 hr with the second antibody goat anti‐mouse IgG (Alexa Fluor^® 594, cat. no. A32740, Invitrogen) and counterstained with 0.5 mg/ml of DAPI solution for 10 min at room temperature. The localization of NF‐κB was imaged using a CARV confocal imager (BD Biosciences) as described previously (Chen, Tillberg, & Boyden, 2015).