P iκbαser32 36

The MEK/ERK inhibitor U0126, PI3K/AKT inhibitor LY294002, NF‐κB inhibitor BAY11‐7082, mTOR inhibitor rapamycin, and HIF‐1α inhibitor PX‐478 were obtained from Selleck Chemicals (Houston, TX, USA. The primary Abs against EGFR [EP38Y] (ab52894), p‐ERK1/2, pT202/pT204) (ab50011), ERK1/2 (ab17942), HIF‐1α (ab51608), PD‐L1 (ab205921), His tag (ab18184), and Actin (ab8226) were purchased from Abcam (Cambridge, UK), and Abs against p‐AKT (Ser473) (#4060), AKT (#4691), p‐S6 (Ser235/236) (#4858), S6 (#2217), and p‐IκBα (Ser32/36) (#9246) were from Cell Signaling Technology (Beverly, MA, USA). Additionally, two primary Abs used for flow cytometry analysis, PE‐PD‐L1 (557924) and APC‐EGFR (563577), and their respective isotype control Abs, PE Mouse IgG1 (κ isotype control, 555749) and APC Mouse IgG2b (κ Isotype control, 557903), were obtained from BD Biosciences (San Jose, CA, USA).

The protein levels in HepG2 cell homogenates were measured using a standard Western blotting protocol as previously described [7 (link)]. The protein concentrations of the samples were determined with the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein from each sample were mixed with loading buffer and separated on SDS-PAGE gels by electrophoresis. The proteins were then transferred onto a polyvinylidene fluoride membrane (GE Healthcare BioSciences, Piscataway, NJ, USA), and the membranes were incubated overnight at 4℃ with a 1:2,000 dilution of antibodies to p-NF-κB-p65 (Ser536, Cell Signaling Technology, Danvers, MA, USA), t-NF-κB (p65, Cell Signaling Technology), p-inhibitor of NF-κBα (p-IκBα, Ser32/36, Cell Signaling Technology), and t-IκBα (Santa Cruz Biotechnology). Next, the membranes were incubated with peroxidase-conjugated secondary antibodies, and the signals were visualized with an enhanced chemiluminescence system detection reagent (GE Healthcare Bio-Sciences). Positive immunoreactive bands were quantified with a densitometer (LAS-3000, FUJIFILM, Tokyo, Japan) and normalized to the levels of total protein and β-tubulin (Santa Cruz Biotechnology).

AS-IV (Cat.83207-58-3, and HPLC purity >98%): Tauto Biotech (Shanghai, China). LPS (E.coli 0111:B4) (Cat.437620), 3,3′-diaminobenzidine (DAB) (Cat.D8001), thioflavin-S (Cat.T189) and BCA protein assay reagents (Cat.BCA1): Sigma (China). Anti-Iba1 primary antibody (Cat.012-26723): Wako (Japan). Biotin-rabbit anti-mouse IgG (Cat.7074): Life Technologies (USA). Streptavidin conjugated-horseradish peroxidase (Cat.RABHRP3) and TRIzol: Invitrogen (USA). p-P65 (Ser536) (Cat.3033), p-IκBα (Ser32/36) (Cat.9246), AF488 fluorescent antibody (Cat.15008) and corresponding antibodies against total proteins: Cell Signaling Technology (Shanghai, China). Protein and nucleic acid precipitation solution (Cat.A7124) (Cat.A7943): Promega Biotech (China). Agarose (Cat.A9539): Biowest (France). Nitric oxide (NO) assay kit (Cat.S0025), RIPA and CCK8 kit: Beyotime (China). Reverse transcriptase (Cat.2690A): Takara (China). SYBR selection master mix: Life Technologies (USA). DMEM culture medium, fetal bovine serum: Gibco (USA).