pparα  (Proteintech)

Journal: Gut

Article Title: Gut microbe alleviates stress-related cancer metastasis by oleic acid degradation

doi: 10.1136/gutjnl-2025-335627

Figure Lengend Snippet: Bifidobacterium animalis and BL21-ohyA can counteract stress-induced tumour metastasis. ( A ) Schematic illustration of the in vitro migration assay. MC38 cells were treated with DMSO or OA for 48 hours and subjected to Transwell assay. ( B ) Representative Transwell assay images and quantification of relative optical density (n=3). ( C ) Schematic of the metastatic CRC mouse model. Mice were orally gavaged with PBS, OA or OA plus B. animalis (n=4 per group). ( D ) Representative IVIS images and quantification of lung metastases by total flux. ( E ) Representative images and quantification of lung weight in different groups. ( F ) PCA of transcriptome analysis from MC38 and 4T1 cells treated with OA. ( G ) KEGG enrichment analyses of differentially regulated genes in MC38 and 4T1 cells. ( H ) Schematic diagram of PPAR signalling pathway activation by OA and its downstream effects. ( I ) Schematic of the metastatic CRC mouse model treated with FH535 (PPAR antagonist) or GW9662 (PPARγ antagonist) (n=4 per group). ( J ) Representative IVIS images and quantification of lung metastases. ( K ) Representative images and quantification of lung weight. ( L ) Genome map of B. animalis , highlighting the location of oleate hydratase ( ohyA ) gene. ( M ) Quantification of OA levels in supernatant of E. coli or B. animalis (n=3 per group). ( N ) SDS-PAGE gel showing expression of OhyA in engineered BL21 strains. ( O ) Quantification of OA levels in supernatant of BL21-vector or BL21-ohyA (n=3 per group). ( P ) Schematic of metastatic CRC model under CRS after gavaged with BL21-vector or BL21-ohyA (n=5 per group). ( Q ) Representative IVIS images and quantification of lung metastases. ( R ) Representative images and quantification of lung weight. ( S ) Quantification of faecal OA levels in CRS mice gavaged with BL21-vector or BL21-ohyA . ( T ) Schematic of metastatic CRC model with OA feeding. Mice were gavaged with BL21-vector or BL21-ohyA (n=4 per group). ( U ) Representative IVIS images and quantification of lung metastases. ( V ) Representative images and quantification of lung weight. ( W ) Quantification of faecal OA levels in mice gavaged with BL21-vector or BL21-ohyA . Data are presented as mean±SEM. Two-group comparisons were analysed by unpaired two-tailed Student’s t-test, and multiple-group comparisons by one-way ANOVA with Tukey’s post hoc test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; CRC, colorectal cancer; CRS, chronic restraint stress; DMSO, dimethyl sulfoxide; OA, oleic acid; PBS, PBS, phosphate buffered saline; PCA, principal component analysis.

Article Snippet: The sections are then incubated with the anti-PPAR alpha antibody (MCE, HY- P80871 ), anti-PPAR delta + beta antibody (Bioss, bs-0250R) or anti-PPAR gamma antibody (Proteintech, 16643-1-AP) at 4°C overnight.

Techniques: In Vitro, Migration, Transwell Assay, Activation Assay, SDS Page, Expressing, Plasmid Preparation, Two Tailed Test, Saline

Journal: Bioactive Materials

Article Title: A foam cell-targeted lipophagy restoration strategy stabilizes vulnerable atherosclerotic plaques

doi: 10.1016/j.bioactmat.2026.02.041

Figure Lengend Snippet: Schematic of the anti-atherosclerotic mechanism of OPN-HMCN@MLT. ( A ) The study commenced with the synthesis of mesoporous carbon nanospheres (MCN) functionalized with an OPN-binding peptide and hyaluronic acid to construct the OPN-HMCN nanoplatform. The OPN-binding peptide was designed to recognize OPN enriched in the extracellular matrix and on the surface of foam cells, thereby enabling selective accumulation in OPN-rich pathological regions. Following OPN recognition, OPN-HMCN@MLT undergoes CD44-dependent endocytosis. Melatonin (MLT), a lipid autophagy–promoting agent, was subsequently encapsulated within the nanocarrier to form OPN-HMCN@MLT. Firstly, the released MLT can bind to and upregulate the expression of PPARα and PPARγ, which then promote the expression of downstream genes (ABCA1, ABCG1, ACOX-1, and CTP1A) and trigger the lipophagy. ( B ) Subsequently, its lipophagy-enhancing effects, including ABCA1/G1-mediated cholesterol efflux and CTP1A/ACOX-1-mediated mitochondrial fatty acid oxidation, were studied to confirm the reversal of foam cell formation. ( C ) These effects eventually promote foam cells to reverse into macrophages. Abbreviations: MCN, mesoporous carbon nanoparticle; OPN, osteopontin; MLT, melatonin; LDL, low-density lipoprotein; ox-LDL, oxidized low-density lipoprotein; PA, Photoacoustic.

Article Snippet: To block nonspecific binding, membranes were incubated with 5% skim milk for 1 h. Thereafter, membranes were incubated overnight at 4 °C with primary antibodies against ABCA1, ABCG1, ACOX1, CPT1A, LC3 (ab192890, 1:2000, abcam), LAMP1 (84658-5-RR, 1:8000, Proteintech), PPARα (66826-1-Ig, 1:3000, Proteintech), PPARγ (66936-1-Ig, 1:10000, Proteintech), P62 (18420-1-AP, 1:10000, Proteintech), MCAD (55210-1-AP, 1:3000, Proteintech), LCAD (17526-1-AP, 1:10000, Proteintech), tubulin (80762-1-RR, 1:10000, Proteintech), GAPDH (60004-1-Ig, 1:50000, Proteintech), and β-actin (66009-1-Ig, 1:20000, Proteintech).

Techniques: Binding Assay, Construct, Expressing

Journal: bioRxiv

Article Title: Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration

doi: 10.64898/2026.05.07.723151

Figure Lengend Snippet: A) Pull-down of PPAR-alpha is performed to assess its ubiquitination profile after BPA administration. PPAR-alpha antibody and its isotype control IgG antibody were used for pulling PPAR-alpha from the protein lysate. The PPAR-alpha enriched samples were immunoblotted against anti-ubiquitin and anti-PPAR-alpha antibody, along with the input samples from un-enriched fractions. The red highlighted region indicates the mono-ubiquitination of PPAR-alpha in BPA samples. B ) Individual structures of PPAR-alpha-LBD from the AlphaFold model (ice-blue) and the crystal structure PDB 8HUK (orange) are shown, with the 242–267 loop region circled. The AlphaFold model represents the dominant conformation observed in most ligand-bound PPAR-alpha crystal structures, whereas the 8HUK structure displays the loop in an open conformation. ( C ) Superimposition of the AlphaFold and 8HUK structures reveals positional shifts of the 242-267 loop. The docked pose of BPA (green) and the key lysine residues K252, K257, K266, and K349 are highlighted to illustrate their spatial relationship to the ligand-binding pocket. ( D ) Close-up views of the lysine side chains show their orientation within the dynamic loop region and proximity to the BPA binding site. E ) Transcript abundance of UCHL1, USP5, MuRF1, HUWE1, and TRIM46 was measured utilizing qRT-PCR and normalized to GAPDH expression using the ΔΔCt method. Results are represented as mean ± SEM from three independent biological replicates. Multiple students’ t-test was employed, considering p-value < 0.05 as significant. F) Western blot analysis of PPAR-alpha, MuRF1, TRIM46, USP5, and UCHL1 from three biological replicates of BPA-exposed and control samples. GAPDH is used as a loading control for the respective experiments. G) The densitometric analysis of acquired immunoblots is summarized as mean ± SEM (N=3). The statistical significance was evaluated using multiple Student’s t-tests. The differences were considered significant at p < 0.05.

Article Snippet: Molecular dynamics stimulation reveals a novel mechanism by which BPA directly binds to PPAR-alpha, stabilizing a flexible lysine-rich loop and exposing K252, K257, K266, and K349 residues, thereby promoting mono-ubiquitination of PPAR-alpha.

Techniques: Ubiquitin Proteomics, Control, Ligand Binding Assay, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration

doi: 10.64898/2026.05.07.723151

Figure Lengend Snippet: A) Subcellular distribution of ubiquitinated PPAR-alpha in BPA-treated HTR8/SVneo cells visualized using confocal microscopy. Cells were immune-stained with anti-PPAR-alpha (green) and anti-ubiquitin (red) antibodies. While for the nucleus, the cells were counter-stained using DAPI (blue). Under basal conditions, ubiquitinated PPAR-alpha localizes primarily in the cytosol, and upon BPA exposure, it translocates to the nucleus. Images were captured using a Leica SP8 confocal microscope with a 63X oil objective. Scale bars = 10 μm. B) STRING-enriched protein-protein interaction network of 41 unique target genes of PPAR-alpha highlights the proteins involved in migration, curated from literature. Node colours demonstrate the association of genes with different migration phenotypes mentioned below the network. C) qRT-PCR was performed using gene-specific primers for TGFβ1, CPT1A, CYP1A1, CITED2, HMGCS1, NFKBIA, STAT5A, and CGB3/CGB5 and normalized to GAPDH utilizing the ΔΔCt method. Each data points reflects the mean ± standard error of the mean (SEM) from three independent biological replicates. Statistical analysis was performed using Student’s t-test and significant differences were considered at p < 0.05.

Article Snippet: Molecular dynamics stimulation reveals a novel mechanism by which BPA directly binds to PPAR-alpha, stabilizing a flexible lysine-rich loop and exposing K252, K257, K266, and K349 residues, thereby promoting mono-ubiquitination of PPAR-alpha.

Techniques: Confocal Microscopy, Staining, Ubiquitin Proteomics, Microscopy, Migration, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration

doi: 10.64898/2026.05.07.723151

Figure Lengend Snippet: The image depicts mechanistic insights of BPA-mediated PPAR-alpha ubiquitination via direct BPA binding, which stabilizes the K252, K257, and K266 residues or indirect regulation of E3 ligase MuRF1 and DUBs USP5. Both facilitate the build-up of mono-ubiquitinated PPAR-alpha upon BPA exposure. This ubiquitinated PPAR-alpha localizes to the nucleus and dysregulates key migration-associated target genes, which ultimately contribute towards reduced migration phenotype in HTR8/SVneo cells.

Article Snippet: Molecular dynamics stimulation reveals a novel mechanism by which BPA directly binds to PPAR-alpha, stabilizing a flexible lysine-rich loop and exposing K252, K257, K266, and K349 residues, thereby promoting mono-ubiquitination of PPAR-alpha.

Techniques: Ubiquitin Proteomics, Binding Assay, Migration

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: RNA-seq identifies ABCC5 as a potential key downstream effector of PPARγ in HS. (A) Volcano plot illustrating differentially expressed genes between the WT + HS and PPARγ-OE + HS groups. (B) GO enrichment analysis of differentially expressed genes between the WT + HS and PPARγ-OE + HS groups. (C) KEGG pathway enrichment analysis of DEGs between the WT + HS and PPARγ-OE + HS groups. (D) Heatmap displaying expression changes of ABC transporter family members across the indicated groups. (E) Measurement of cellular free fatty acids and triglycerides in cells under the indicated treatments. (F) RT-qPCR analysis of PPARγ mRNA expression in PPARγ NC + HS and PPARγ OE + HS cells. (G) RT-qPCR analysis of selected ABC transporter genes (ABCC5, ABCB1A, ABCC6, TAP2, ABCA6, ABCB4, ABCC10, ABCA2, ABCG4, ABCA1, ABCA8A, ABCA9, ABCB2, ABCB7, and ABCA3) under the specified conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the PPARγ-NC + HS group (E–G). Statistical comparisons were performed using Student's t-test (F–G) or one-way ANOVA (E).

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: Time-dependent changes in ABCC5 expression in vivo. (A) Representative immunofluorescence images of ABCC5 (green) and DAPI (blue) in cardiac tissues from sham mice and from mice subjected to HS at the indicated time points after injury. (B) Representative immunohistochemical staining of ABCC5 in cardiac tissues from sham and HS-injured mice. (C) RT-qPCR analysis of Leptin mRNA in cardiac tissues after 2.5 h or 3 weeks of heat injury. (D) Representative immunofluorescence images of ABCC5 in cardiac sections from PPARγ-cKO mice after HS). (E–F) Representative immunofluorescence images of PPARγ and ABCC5 in cardiac sections from PPARγ-cKO mice at 3 weeks after HS). Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the sham group (B–C). Statistical comparisons were performed using Student's t-test (B) or one-way ANOVA (C).

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Expressing, In Vivo, Immunofluorescence, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: ABCC5 siRNA abolishes the cardioprotective effects of PPARγ overexpression against HS ​. (A) Luciferase activity in cells co-transfected with ABCC5 wild-type or mutant (Mut1/2/3) reporter plasmids and adenovirus expressing PPARγ. (B) CUT&Tag assay using a PPARγ-specific antibody to detect PPARγ binding to the ABCC5 promoter. (C) RT-qPCR analysis of ABCC5 mRNA in cells transfected with control siRNA or ABCC5 siRNA. (D – F) Cell morphology and viability in cells transfected with ABCC5 siRNA and/or PPARγ overexpression vector under HS conditions. (G – H) Apoptosis levels measured by flow cytometry in cells transfected with ABCC5 siRNA and PPARγ-OE under HS conditions. (I – J) DCFH-DA staining for ROS detection in cells transfected with ABCC5 siRNA and PPARγ-OE under HS conditions. (K – L) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (M) Western blot analysis of PPARγ, ABCC5, ABCC1, Leptin, and β-actin (loading control) in cells treated as follows: PPARγ-NC + HS, PPARγ-OE + HS, and PPARγ-OE + ABCC5 siRNA + HS. Molecular weight markers are shown on the right. (N) Quantification of protein levels normalized to β-actin, corresponding to the blots in (M). Data are presented as mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 versus the indicated control, PPARγ + ABCC5 group (A–B), control siRNA group (C), PPARγ-NC + HS group, PPARγ-OE + HS group, or PPARγ-OE + ABCC5 siRNA + HS group (D–L), or versus the PPARγ-NC + HS group and PPARγ-OE + HS group (M − N). Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing, Binding Assay, Quantitative RT-PCR, Control, Plasmid Preparation, Flow Cytometry, Staining, Membrane, Fluorescence, Western Blot, Molecular Weight

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The PPARγ/ABCC5 pathway alleviates lipid accumulation in HS-injured mice ​. (A – D) Cardiac sections from sham mice and from mice at indicated time points after HS were stained with HE (A) , PSR (B) , Masson's trichrome (C) , or Oil Red O (D) (n = 3 per group). (E) Serum levels of HDL-C and LDL-C in sham mice and in mice 3 weeks after HS (n = 6–7 per group). Error bars represent mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the sham group. Statistical comparisons were performed using Student's t-test.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Staining

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: Rosiglitazone pretreatment alleviates HS-induced myocardial injury via the PPARγ/ABCC5 pathway in HL-1 cells ​. (A – C) Cell viability and morphology in cells treated with different concentrations of rosiglitazone (5 μM, 10 μM, 20 μM, 40 μM) under HS conditions. (D – E) Apoptosis levels in cells treated with different concentrations of rosiglitazone under HS conditions. (F–I) DHE staining (F) and DCFH-DA staining (I) for ROS detection in cells treated with different concentrations of rosiglitazone under HS conditions. (J – K) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (L) RT-qPCR analysis of PPARγ, ABCC5, Leptin, and SREBP-1c in cells treated with different concentrations of rosiglitazone under HS conditions. (M – N) Representative Western blots and quantification of PPARγ, ABCC5, ABCC1, ABCG1, ABCA1, and Leptin in cells treated with different concentrations of rosiglitazone under HS conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the control group or the HS group. Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Staining, Membrane, Fluorescence, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The PPARγ agonist rosiglitazone confers pharmacological protection against HS-induced myocardial dysfunction ​. (A – C) Cell viability and morphology in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (D – E) Apoptosis levels measured by flow cytometry in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (F) LDH release in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (G – H) DCFH-DA staining for ROS detection in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (I – J) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (K) RT-qPCR analysis of PPARγ and CPT1β mRNA in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (L) Representative Western blots and quantification of PPARγ, ABCC5, PGC-1α, and PPARγ in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the control group, the HS group, or the ROSI + HS group as indicated. Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Transfection, Flow Cytometry, Staining, Membrane, Fluorescence, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The proposed scheme describing the signaling pathway of PPARγ/ABCC5-elicted cardioprotective effect against HS.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: