Ab76302

For Western blotting, total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Atlanta, GA, USA) [17 (link)]. After washing and blocking, the membranes were incubated with primary antibodies against VacA (sc-32,746, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500), p65 NF-κB (ab32536, 1:5000), p-p65 NF-κB (ab76302, 1:1000), and GAPDH (ab8245; Abcam, 1:10,000). After washing again, the membranes were incubated with secondary antibody (ab205718 and ab6728; Abcam, 1:10,000) for 2 h at 25°C. Protein bands were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).

Western blot analysis was performed as described by Rong et al. [30 (link)]. The basic steps included protein concentration, polyacrylamide gel electrophoresis, film rotation, closing, primary antibody incubation, film washing, secondary antibody incubation, film washing, and enhanced chemiluminescence (ECL) development. The antibodies were: anti-TGF-β1 (ab215715, Abcam), anti-α-SMA (ab7817, Abcam), anti-Smad2 (ab40855, Abcam), anti-Smad3 (ab40854, Abcam), anti-Smad7 (25840-1-AP, Proteintech), anti-PI3K (ab191606, Abcam), anti-p-Akt^ser473 (66444-1-Ig, Proteintech), anti-mTOR (ab134903, Abcam), anti-p-IkBα (ab133462, Abcam), anti-p-IKKβ (AF3010, Affinity), and anti-p-P65 (ab76302, Abcam).

Proteins in the brain tissues and lung tissues were harvested by a RIPA buffer (P0013D, Beyotime, China) and their concentrations were qualified with a BCA Kit (pc0020, Solarbio, China). After denaturation, the protein samples were separated by electrophoresis. Proteins in the gel were transferred to a nitrocellulose membrane (10600023, GE Healthcare Life, USA), which was then sealed a 5% skim-milk. After that, they were reacted with primary antibodies at 4 °C overnight. After washing, they were reacted with anti-rabbit HRP (1:5000, #7074, CST, USA) at 37 °C for 1 h. In the end, the protein signals were developed by the ECL reagent (35055, Pierce, USA) in a gel imaging system (A44114, Invitrogen, USA). The primary antibodies of TNF-α (ab205587, 1:1000), IL-1β (ab254360, 1:1000), p-NF-κB (ab76302, 1:1000), NF-κB (1:5000, ab32536), p-IKBα (1:10,000, ab133462), IKBα (1:5000, ab32518), HIF-1α (1:1000, ab179483), and GAPDH (1:5000, ab199554) were obtained from Abcam (UK).

The cells were lysed for 20 min in 180 μL of lysis buffer (Beyotime, Shanghai, China) on ice before the centrifugation at 4 °C and 14,000 rpm for 10 min. The total protein concentration was estimated using a bicinchoninic acid (BCA) protein detection kit (CWBIO, Shanghai, China) with bovine serum albumin (BSA) as the standard. The proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After being blocked with 5% (w/v) skimmed milk for 2 h, the membranes were probed with corresponding primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h. The primary antibodies used were: TLR4 (1:1000, Abcam, ab13556), myeloid differentiation factor 88 (MyD88, 1:1000, Abcam, ab133739), nuclear factor kappa-B (NF-κB) p65 (1:1000, Abcam, ab32536), NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), claudin-1 (1:2000, Abcam, ab211737), occludin (1:1000, Abcam, ab216327), ZO-1 (1:1000, Abcam, ab276131), myosin light-chain kinase (MLCK, 1:1000, ABclonal, A3835), and β-actin (1:5000, Abcam, ab179467). The protein intensity was quantified using the Image J software (version 1.8.0, NIH, Bethesda, MD, USA).

The kidney tissue was ground in liquid nitrogen and cleaved with a strong RIPA buffer (EA0002, Sparkjade). The protein concentration was determined using a bicinchoninic acid (BCA) protein quantitation kit (EA0002, Sparkjade). Primary antibodies targeting NF-κB p65 (ab19870, Abcam), NF-κB p65 (phospho-S536) (ab76302, Abcam), and beta-actin (20536-I-AP, Proteintech) were incubated overnight with the target protein at 4 °C. The samples were then incubated with HRP-conjugated secondary antibodies (EF0002, Sparkjade). Protein expression was measured using an enhanced chemiluminescence reagent (ECL) kit (ED0015, Sparkjade).

An HFHSD (23.3% protein, 46% carbohydrate, and 20.4% fat) was purchased from Research Diets Company, D12327 (New Brunswick, NJ, USA).
Glucosamine (GLC; D-glucosamine hydrochloride, biotech grade, CAS number: 66-84-2) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China).
Metformin was purchased from Sino-US Shanghai Squibb Pharmaceutical Co., Ltd. (CAS number: ABH5474) (Shanghai, China).
The primary antibodies used were an anti-TLR4 monoclonal antibody (66350-1-Ig, 1:1000), anti-CD14 monoclonal antibody (60253-1-Ig, 1:1000), anti-MYD88 monoclonal antibody (67969-1-Ig, 1:1000), anti-GAPDH polyclonal antibody (10494-1-AP, 1:5000), anti-ZO-1 polyclonal antibody (21773-1-AP, 1:100), anti-Occludin polyclonal antibody (27260-1-AP, 1:100), and anti-Claudin-1 polyclonal antibody (13050-1-AP, 1:500). The above antibodies were purchased from Proteintech (Wuhan, China). An anti-NF-κB p65 polyclonal antibody (ab16502, 1:1000) and anti-NF-κB p65 (phosphor-S536) polyclonal antibody (ab76302, 1:1000) were purchased from Abcam Company (Cambridge, UK).

Western blotting was performed using the standard SDS-PAGE separation technique as previously reported^{18 (link),19} . The harvested cells were disrupted with RIPA cleavage buffer (Cell Signaling Technology). Proteins were extracted from exosomes using a Total Exosome Protein Isolation Kit (Invitrogen, Carlsbad, CA, USA) following the instructions supplied. The lysates were collected after centrifugation, and the protein concentration was quantified with BCA kit (Thermo Fisher Scientific). 50 mg of protein was added to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with antibodies against phosphor-JNK (ab124956), phospho-MAPK (ab195049), phospho-ERK (ab201015), phospho-NF-κB (ab76302), TRAF-6 (ab33915) and GAPDH (ab8245) purchased from Abcam. Subsequently, the membranes were incubated with a secondary antibody for 1.5 h at room temperature. Immunoreactive protein bands were detected using an Odyssey scanning system (USA), quantified by Image J, and normalized to the corresponding amount of total protein. The experiment was repeated in triplicate with 3 replicates.