Total p65

Cells were lysed in RIPA buffer with added protease inhibitors and protein content was measured by BCA assay. Total protein was resolved using SDS-PAGE and subjected to immunoblot analysis. Nuclear and cytoplasmic protein fractions were separately processed similar to total protein and analyzed by immunoblot. Densitometric analyses were performed using ImageJ and phospho-p65 values were normalized to total p65. Uncropped blots are included with supplemental data. Antibodies used were phospho-STAT3 (1:1000, Cell Signaling #9131S), phospho-STAT5 (1:1000, Cell Signaling 9314S), phospho-p65 (1:1000, Cell Signaling #3033S), total STAT3 (1:1000, Cell Signaling #12640), total STAT5 (1:1000, Cell Signaling #94205), total p65 (1:1000, Cell Signaling #8242), GAPDH (1:10,000, Cell Signaling #2118), β-tubulin (1:1000, Cell Signaling #2146S), Lamin B1 (1:1000, Cell Signaling #12586S). We detected two bands for total STAT5 proteins, which correspond to Stat5a and STAT5b, isoforms of STAT5 encoded by homologous genes^{24 (link)}. We also detected two bands for p65, which may correspond to one of the reported isoforms for p65, of which the larger one is transcriptionally active^{25 (link)}.

Total protein was obtained using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and quantified with a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Next, the proteins in the lysates were separated by SDS-PAGE gels and transferred to PVDF membranes (GE Healthcare) followed by incubation in a 5% skim milk solution for 1 h at room temperature. Subsequently, the specific primary antibodies were incubated in the membranes at 4°C overnight, including HMGB1 (#6893, 1:1,000 dilution), TLR4 (#14358, 1:1,000 dilution), Myd88 (#4238, 1:1,000 dilution) IκB-α (#4812, 1:1,000 dilution), phospho-IκB-α (#2859, 1:1,000 dilution), phospho-NF-κB p65 (#3033, 1:1,000 dilution), total p65 (#8245, 1:1,000 dilution) and β-actin (#3700, 1:1,000 dilution) (Cell Signaling Technology). Subsequently, the corresponding anti-rabbit secondary antibodies (cat no.3678, 1:2,000) were added into the membranes for 2 h at room temperature. The protein band was detected by chemiluminescence with Pierce ECL kits (Millipore). Semi-quantification was performed using ImageJ version 1.46 (MD, USA). The original images of Western Blot are uploaded as Supplementary Material.

The cellular proteins were extracted by lysing cells in a RIPA buffer containing 1mM Phenylmethylsulfonyl Fluoride (PMSF). The protein samples were separated by SDS-PAGE electrophoresis, and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with bovine serum albumin (5%) for 1 h at room temperature under slight shaking, the membranes were incubated with primary antibodies (1:500) at 4 °C for 12 h and then blotted with secondary antibody for 2 h at room temperature. Then the bands were stained with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA). In this study, the cells including M2-M and TAMs were treated with 50 μg/mL IAPS-2 for 24 h, then the expression of phosphorylated P65 (p-P65), total P65, phosphorylated STAT1 (p-STAT2), total STAT1 (T-STAT1), phosphorylated STAT3 (p-STAT3) and total STAT3 (T-STAT3) in both cells were evaluated by Western blot. The corresponding antibodies, including phosphor-P65, Total-P65, phosphor-STAT1, Total-STAT1, phosphor-STAT3, Total-STAT3 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

Livers were collected from sickle mice with DSFCs after 4 h of reoxygenation. The livers were flash-frozen in liquid N₂ and stored at -85°C until use. Microsomes and nuclear extracts were isolated from the livers of mice as previously described [20 (link)]. Immunoblots of cellular subfractions were immunostained with primary antibodies to Nrf2 (Proteintech #16396-1-AP), HO-1 (Enzo #ADI-OSA-111), NF-ĸB phospho-p65 (Ser536, Cell Signaling #3031), total p65 (Cell Signaling #3034), VCAM-1 (Abcam #ab174279) and GAPDH (Sigma Aldrich #G9545). Primary antibodies were labeled with the appropriate secondary antibodies conjugated to alkaline phosphatase and visualized with ECF substrate (GE Healthcare) and a Typhoon FLA 9500 scanner (GE Healthcare). Immunoreactive bands on images were quantitated using ImageJ software (NIH). Mean relative expression of protein bands from animals treated with vehicle or HBI-002 groups were calculated.

Liver tissues were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) on ice to prepare for total protein extraction. Total protein extractions were denatured with SDS loading buffer by heating at 95°C for 5 min. After blocking with 5% non-fat milk in tris-buffered saline tween (TBST), the membranes were incubated separately with total JNK (Cell Signaling Technology, MA, United States), p-JNK (Cell Signaling Technology, MA, United States), total p65 (Cell Signaling Technology, MA, United States), p-p65 (Cell Signaling Technology, MA, United States), total p38 (Cell Signaling Technology, MA, United States), p-p38 (Cell Signaling Technology, MA, United States), total ERK (Cell Signaling Technology, MA, United States), and p-ERK (Cell Signaling Technology, MA, United States) antibodies overnight at 4°C. Finally, the protein bands were incubated with secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature and were then visualized using an enhanced chemiluminescence (ECL) detection kit (Vazyme, Wuhan, China).