Ripa lysate

BMMs and HUVECs were seeded in 6‐well plates (1×10⁵ cells per well) and treated with a medium containing 50 µm PAP or PDAP NPs. The cells were lysed on ice for 0.5 h using RIPA lysate (Servicebio Co., Ltd., China) containing protease inhibitors and the supernatant was collected by centrifugation. After the supernatants were separated by SDS‐PAGE gel electrophoresis, they were transferred to PVDF membranes (Millipore, Billerica, MA) which were then blocked for 1 h with 5% BSA (Servicebio Co., Ltd., China) at room temperature and incubated overnight at 4 °C with the primary antibodies, including anti‐HIF‐1α (Servicebio Co. Ltd., China), anti‐VEGF (Servicebio Co., Ltd., China), anti‐TRAP (Abcam, UK), anti‐CTSK (Abcam, UK), anti‐NFATc1 (Cell Signaling Technology, Beverly, MA), anti‐HO‐1 (Servicebio Co., Ltd., China), and anti‐β‐actin (Servicebio Co. Ltd., China). After incubation with the corresponding secondary antibodies (Servicebio Co., Ltd., China) for 1 h, the blots were detected by a chemiluminescence system and the optical density values were measured with Image J software.

Cells were washed three times with pre-cooled PBS and analyzed using a high strength RIPA lysate (Servicebio, Wuhan, China) on ice after adding a protease inhibitor (Servicebio, Wuhan, China). Lysates were collected and centrifuged at 12,000× g for 5 min at 4 °C. The protein concentration was determined using the BCA protein concentration assay kit (Beyotime, Shanghai, China) and stored at −20 °C.

Cells and subcutaneous xenograft tumor samples were lysed with RIPA lysate (G2002, Servicebio, Wuhan, China) to extract total protein after treatment with Tim-AIII. The protein samples were loaded onto 4% or 10% or 12% sodium dodecyl sulfate-polyacrylamidegel electrophoresis gels (SDS-PAGE) gels and then transferred to a 0.45 μm PVDF membrane. PVDF membrane was immersed in TBST containing 5% skim milk and blocked at RT on a shaker for 1.5 h, then incubated with specific primary antibodies on a shaker overnight at 4 ° C. The PVDF membrane was immersed in the secondary antibody incubation solution for 1.5 h after being rinsed with TBST three times, 10 min for each time. After incubation with the ELC reagent (a mixture of solution A and B in equal proportion), the membranes were visualized by imaging system (iBright 750, Invitrogen, USA).

The frozen tissue was removed, and RIPA lysate (Servicebio Technology Co., Ltd, Wuhan, China), protease inhibitor, and phosphorylated protease inhibitor (Boster Biological Technology Co., Ltd. Wuhan, China) were added, and the tissue was homogenated. After centrifugation, the supernatant was extracted to prepare protein samples. Nuclear protein was extracted according to the instructions of the kit (Beyotime Biotechnology Co., Ltd. Shanghai, China) and used to detect the expression of nuclear SREBP-1c. The equivalent protein sample or nuclear protein sample was added to 8% sodium dodecyl sulfate-polyacrylamide gel to undergo electrophoresis, and the protein was then transferred to the polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skimmed milk powder, the PVDF membrane was incubated overnight with the following antibodies: p-MYPT-1, MYPT-1, p-AMPK, AMPK, LDLR, SREBP-1c, FAS, or GAPDH at 4 °C. After cleaning the primary antibody, the membranes were incubated with the corresponding secondary antibody and then treated with the Super ECL detection reagent (Yeasen Biotechnology Co., Ltd. Shanghai, China). The obtained bands were analyzed using Vision Capt software (Kunming Beijie Technology Co., Ltd. Kunming, China).

First, 6 mice were selected from each group, and 30 mg of brain tissue from each of the mice was extracted and added to 300 μL of a mixture of RIPA lysate (Servicebio, Wuhan, China) and 3 μL of cocktail protein inhibitor (CME, Shanghai, China) for homogenization and centrifugation. The supernatant was obtained, followed by the estimation of protein concentration using a BCA protein concentration assay kit (Beyotime, Shanghai, China). The samples were then sequentially sampled, electrophoresed, transferred to membranes, sealed, and incubated with primary antibodies for ZO−1 (1:1000, Proteintech, Wuhan, China), occludin (1:1000, abcam, Cambridge, UK), claudin−5 (1:1000, abcam, Cambridge, UK), and β-actin (1:50,000, abclonal, Wuhan, China) overnight at 4 °C. The next day, the samples were washed with PBST for 3 × 10 min, incubated with secondary antibodies (1:1000, Proteintech, Wuhan, China), and shaken at room temperature for 1 h 20 min. The samples were washed with PBST for 3 × 10 min and imaged using the ECL luminescence kit (Servicebio, Wuhan, China) in a Tanon-5200 gel system to capture pictures. The integrated grayscale values of the samples were analyzed using ImageJ software, and the relative protein expression levels were quantified.

Renal tissues and cells were lysed using RIPA lysate (Servicebio). The BCA method was used to quantify total protein. Proteins were separated on polyacrylamide gels , then moved to a polyvinylidene fluoride membrane 5% skim milk was used to prevent non-specific binding to the membrane for 2 h at room temperature, followed by incubation overnight with one of the following primary antibodies: EZH2 (5246S; 98 kDa; 1:1,000; CST), H3K27me3 (9733S; 17 kDa; 1:1,000; CST), H3(17168-1-AP; 17kDa; 1:2000; Proteintech), cleaved-caspase3(19677-1-AP; 17 kDa; 1:1,000; Proteintech), Bcl2 (12789-1-AP; 26 kDa; 1:1,000; Proteintech), Bax (GB114122; 21 kDa; 1:800; Servicebio), MCP-1(GB113239; 11 kDa; 266 1:500; Servicebio), Sox9 (67439-1-Ig; 56 kDa; 1:1000; Proteintech), β-catenin (GB12015; 92 kDa; 1:500; Servicebio), surviving (GB11177; 16 kDa; 1:1,000; Servicebio), GAPDH (GB12002; 37 kDa; 1:1,000; Servicebio) at 4 °C. Horseradish peroxidase-coupled secondary antibodies were incubated for 1 h with the protein bands after being washed by TBST, followed by visualization using an ECL kit (Biosharp, China) on a ChemiDoc MP system (Bio-Rad, Foster City, CA, USA). The relative density of proteins was measured using ImageJ software (1.8.0). All experiments were performed using three replicates.