Ab199

Protein isolation from primary chondrocytes was performed as we described previously¹⁵ . Western blot was performed using a standard protocol^{15 ,21 (link)}. Antibodies against the following proteins were used: rabbit anti-rat RXRα (Abcam, ab125001, dilution 1:1000); rabbit anti-rat ERα (Abcam, ab32063, dilution 1:1200); rabbit anti-rat p-p65 (Abcam, ab28856, dilution 1:2000); rabbit anti-rat p-IκBα (Abcam, ab32518, dilution 1:2,000); rabbit anti-rat HIF-2α (Abcam, ab199, dilution 1:2000); rabbit anti-rat GADPH (R&D, 2275-PC-100, dilution 1:3000).

Cells were lysed in Laemmli buffer, and the protein extract was resolved on 10% or 12% SDS-polyacrylamide gels and transferred to 0.45 μm nitrocellulose membranes. The membranes were then blocked and probed with antibodies against: HIF2α (ab199, Abcam); HIF1α (610959, BD Transduction Laboratories, Franklin Lakes, NJ, USA); β-actin (A3854, Sigma, Saint Louis, MO, USA). Antibody binding was detected by enhanced chemiluminiscence (Clarity, BioRad, Hercules, CA, USA; and SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, Waltham, MA, USA) and visualized on a digital luminescent image analyzer (Image Quant LAS4000 Mini; GE Healthcare, Chicago, IL, USA).

Western blotting was conducted to quantify the protein expression of hypoxia-inducible factor-1α (HIF-1α, Abcam, AB199) in 4T1 cells. Initially, 4T1 cells were seeded in 6-well plates and incubate overnight. The cells were then divided into three groups: control, Pd@Au-PEG NSs, and Pd@Au-PEG NSs + laser (1064 nm, 0.3 W cm^− 2, with the maximum temperature maintained at 45 °C). Subsequently, the cells were treated with H₂O₂ (100 μM) for a duration of 24 h. To induce hypoxia, the 4T1 cells under different treatments were placed in a hypoxic incubator (Thermo Scientific) maintained at 1.8% O₂ and 5% CO₂ (with the remaining balance being N₂) for a period of 24 h. Cells were harvested and lysed in RIPA buffer containing 1% phosphatase and protease inhibitors, followed by boiling for 10 min at 100 °C. The proteins were subsequently separated using SDS-PAGE electrophoresis and transferred onto Immobilon PVDF membranes (Millipore). The membrane was then blocked with 5% milk in TBST for one hour. The primary antisera against HIF-1α were diluted by 1000 and incubated overnight at 4 °C in the blocking buffer. Subsequently, the membranes were incubated at room temperature for two hours with the secondary antibody. Protein signals were detected using an ECL system (Amersham Biosciences, Buckinghamshire, UK). β-actin were used as internal controls.

Paraffin‐embedded liver biopsy sections (4 µm thick) were immunostained with a primary rabbit antibody against HIF2α (ab199, Abcam) ²⁸ or CD36 (NB400‐144, Novus) diluted to 1:50 and 1:200 respectively, using the DAKO EnVision™+ System (DAKO, Glostrup, Denmark) as described by the manufacturer. Liver tissue area occupied by nuclear HIF2α or CD36‐positive cells was quantified from the images taken using an optical microscope Nikon Eclipse E400 (Nikon). Image analysis procedures were performed with the FIJI software (NIH). Values were obtained in six different lobular areas where hepatocytes are the predominant cell type. The average value was considered as nuclear HIF2α or CD36 expression index for each liver biopsy sample, and expressed as percentage of positive nuclei or arbitrary units respectively, which reflects the intensity of the immunostaining.

Proteins were extracted from snap-frozen kidney cortexes and were quantified with a Bio-Rad protein assay. Equal amounts of protein lysates were loaded and separated on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes or polyvinylidene difluoride membranes (Millipore, USA). Nonspecific proteins were blocked by incubating the membranes in 5% non-fat milk for 1 hour at room temperature. The membranes were then incubated with primary antibodies at 4°C overnight for specific proteins, followed by incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature. HRP activity was visualized using Clarity Western ECL Substrate and a ChemiDoc MP Imaging System (Bio-Rad Laboratories, USA). Image Lab software version 5.1 was applied for densitometric analysis (Bio-Rad Laboratories, USA). The following primary antibodies were used in this study: polyclonal anti-HIF-2α from rabbit (Abcam; ab199; 1:200 dilution), polyclonal anti-HIF-3α from rabbit (Abcam; ab176464; 1:1000 dilution), and monoclonal anti-β-actin from mouse (Sigma; A5441; 1:5000 dilution).