Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 1. The kidney disease risk gene, PPDPF, was up-regulated in the early stages of both human and mouse kidney diseases. (A) Schematic representation of bulk RNA-seq and scRNA-seq on kidney samples of 1d and 5d UUO. (B) DEGs that were significant in UUO 1d but not significant in UUO 5d of bulk RNA-seq. (C) The UMAP of 18 distinct cell types identified by unsupervised clustering. Endo, endothelial cell; podo, podocyte; LOH, loop of Henle; DTL, descending thin limb of Henle’s loop; DCT, distal convoluted tubule; PC, principal cell; IC, intercalated cell; urothelial, urothelial cell; macro, macrophage; B, B lymphocyte; NK, natural killer cell; T, T cell; neutro, neutrophile; DC, dendritic cell; stroma, stroma cell; Immune1, immune cell type 1; Immune2, immune cell type 2. (D) Cell type–specific expression of top DEGs identified from (B). (E) Venn dia- gram exhibits the overlaps among indicated sets (up arrow: up-regulated DEGs). (F) The relative mRNA expression of Ppdpf in the cisplatin (Cis)–, FA-, and UUO-administered kidney samples. (G) Western blots of PPDPF in Cis-, FA-, UUO-, and IRI-administered kidney samples. Eight-week-old male mice were used. NC, negative control. (H) Represen- tative images of double staining of PPDPF and KIM1 in the UUO-administered kidneys. Scale bar, 20 μm. (I) The relative mRNA expression of PPDPF in human AKI kidneys. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (A to D) n = 3, control; n = 3, UUO 1d mice; n = 2, UUO 5d mice. (F) For Cis: n = 6, control; n = 5, Cis-treated mice. For FA: n = 6, control; n = 5, FA-treated mice. For UUO: n = 6, control; n = 6, UUO-treated mice. (G) Three control mice and three diseased mice were included for each model. FC, fold change; GAPDH, glyceraldehyde phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: RNA Sequencing, Expressing, Western Blot, Negative Control, Double Staining, Two Tailed Test, Control

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 5. PPDPF deficiency inhibits NAD+ synthesis through NMNATs. (A) Representative images of double staining of PPDPF and mitochondrial markers ATP5A1, HSP60, or TOMM20 in HK2 cells. Scale bar, 10 μm. (B) Western blots of subcellular fractions of HK2 cells stained with PPDPF, the mitochondrial antibody TOMM20, and the nuclear antibody LAMIN B1. GAPDH was used as internal control. (C) Schematic diagram of PPDPF engages in the synthesis of NAD+ from NMN. (D) NAD+ content of sham and sgRNA-targeted HK2 cells following PBS or NMN treatment. (E) NAD+ content of primary cells isolated from WT and Ppdpf KO kidney following NMN treatment. (F) NAD/ NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 4 hours and measured with SoNar biosensor. (G) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 12 hours and measured with SoNar biosensor. (H) NMNAT activity of primary cells isolated from WT and Ppdpf KO kidney. A450, absorbance at 450 nm. (I) Representative double staining of PPDPF with NMNAT1, NMNAT2, or NMNAT3 in HK2 cells. (J) Representative images of PLA (red). (K) Quantifications of PLA. Scale bar, 10 μm. P value was calculated by two-tailed t test and one-way analysis of variance (ANOVA). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (D) n = 4 for each group. (E) n = 4, WT; n = 4, KO. (F) n = 230, WT; n = 209, KO. (G) n = 87, WT; n = 119, KO. (H) n = 3, WT; n = 3, KO. (J) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Double Staining, Western Blot, Staining, Control, Isolation, Activity Assay, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 7. Ppdpf deficiency exacerbates renal injury in aged mice. (A) Scheme of the experimental process. Kidneys, serum, and urine were harvested at the age of 12 months. (B) Serum creatinine levels in WT and Ppdpf KO mice at 12 months old. (C) Serum BUN levels in WT and Ppdpf KO mice at 12 months old. (D) Serum cystatin C levels in WT and Ppdpf KO mice at 12 months old. (E) Urine albumin levels in WT and Ppdpf KO mice at 12 months old. (F) Representative images of H&E- and Masson- stained kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bar, 20 μm. (G) Quantification of tubular injury score and Masson staining area from (F). (H) Relative mRNA levels of fibrosis markers Col3a1, Col1a1, and Vimentin in kidneys of WT and Ppdpf KO mice at 12 months old. (I) Western blots and quantifications of vimentin, ACTA2, and KIM1 in kidneys of WT and Ppdpf KO mice at 12 months old. (J) Representative images of vimentin and KIM1 staining in kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bars, 20 μm. (K) NAD+ content of whole kidney lysates of WT and Ppdpf KO mice at 12 months old. P value was calculated by two- tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B and C) n = 4, WT; n = 9, KO. (D) n = 3, WT; n = 5, KO. (E) n = 8, WT; n = 8, KO. (F) n = 6, WT; n = 11, KO. (H) n = 7, WT; n = 13, KO. (H) n = 3, WT; n = 3, KO. (K) n = 4, WT; n = 4, KO.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 10. Overexpression of Ppdpf alleviates kidney fibrosis induced by FA administration. (A) Schematic diagram of Ppdpf overexpression in mice. Eight-week-old male mice were in situ injected with AAV9 encoding Ppdpf or scramble. Post–4-week injection, the mice were subjected to FA treatment. (B) The relative expression levels of Ppdpf in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (C) Western blots of Flag and PPDPF in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (D) Western blots of PPDPF in LTL-positive cells from AAV9-Ctrl or AAV9-PPDPF kidneys. (E) Representative images of H&E- and Sirus red–stained kidney sections from AAV9-Ctrl– or AAV9- PPDPF–injected mice following FA administration. Scale bars, 20 μm. (F) Quantifications of tubular injury score and Sirus red staining area from (E). (G) The relative expres- sion levels of fibrosis markers Col3a1, Vimentin, and Fibronectin, as well as injury markers Kim1 and Lcn2 in kidneys of AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. *P < 0.05. (H) Representative images of vimentin, fibronectin, and Col3a1 staining on kidney sections from AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. Scale bars, 50 or 20 μm. (I) NAD+ content of whole kidney lysates from AAV9-Ctrl– or AAV9-PPDPF–injected mice. (J) NMNAT activity of pri- mary cells isolated from AAV9-Ctrl– or AAV9-PPDPF–injected kidneys. P value was calculated by two-tailed t test (B, F, I, and J) and two-way ANOVA (G). P < 0.05 is statisti- cally significant. Data are represented as mean ± SEM. (B) n = 3, AAV9-Ctrl; n = 4, AAV9-PPDPF. (C and D) n = 3 for each group. (F) n = 10 for each group. (G) n = 3, FA-AAV9-Ctrl; n = 4, FA-AAV9-PPDPF. (I) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Over Expression, In Situ, Injection, Expressing, Western Blot, Staining, Activity Assay, Isolation, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 7. Ppdpf deficiency exacerbates renal injury in aged mice. (A) Scheme of the experimental process. Kidneys, serum, and urine were harvested at the age of 12 months. (B) Serum creatinine levels in WT and Ppdpf KO mice at 12 months old. (C) Serum BUN levels in WT and Ppdpf KO mice at 12 months old. (D) Serum cystatin C levels in WT and Ppdpf KO mice at 12 months old. (E) Urine albumin levels in WT and Ppdpf KO mice at 12 months old. (F) Representative images of H&E- and Masson- stained kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bar, 20 μm. (G) Quantification of tubular injury score and Masson staining area from (F). (H) Relative mRNA levels of fibrosis markers Col3a1, Col1a1, and Vimentin in kidneys of WT and Ppdpf KO mice at 12 months old. (I) Western blots and quantifications of vimentin, ACTA2, and KIM1 in kidneys of WT and Ppdpf KO mice at 12 months old. (J) Representative images of vimentin and KIM1 staining in kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bars, 20 μm. (K) NAD+ content of whole kidney lysates of WT and Ppdpf KO mice at 12 months old. P value was calculated by two- tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B and C) n = 4, WT; n = 9, KO. (D) n = 3, WT; n = 5, KO. (E) n = 8, WT; n = 8, KO. (F) n = 6, WT; n = 11, KO. (H) n = 7, WT; n = 13, KO. (H) n = 3, WT; n = 3, KO. (K) n = 4, WT; n = 4, KO.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 8. Ppdpf deficiency exacerbates renal injury in the long-term cisplatin model. (A) Scheme of the experimental approach. Eight-week-old male mice were intra- peritoneally injected with cisplatin (Cis; 7 mg/kg) and euthanized after 28 days. (B) Serum creatinine levels of WT and Ppdpf KO mice with or without cisplatin treatment. (C) Urine albumin levels in WT and Ppdpf KO mice with cisplatin treatment. (D) Representative images of H&E- and Masson-stained kidney sections from WT and Ppdpf KO mice following saline or cisplatin injection. Scale bars, 20 μm. (E) Quantification of tubular injury score and Masson staining area from (D). (F) The relative expression of fibrosis markers Col3a1, Fibronectin, and Vimentin in control or cisplatin-treated WT and Ppdpf KO mice. (G) Western blots of fibrosis markers ACTA2, vimentin, fibronectin, and mitochondrial marker DRP1 in WT and Ppdpf KO mice with or without cisplatin treatment. (H) NAD+ content of whole kidney lysates of WT and Ppdpf KO mice with or without cisplatin treatment. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 4, WT CTR; n = 3, KO CTR; n = 5, WT Cis; n = 5, KO Cis. (C) n = 5, WT Cis; n = 5, KO Cis. (E) n = 3, WT CTR; n = 3, KO CTR; n = 6, WT Cis; n = 6, KO Cis. (F) n = 4, WT CTR; n = 4, KO CTR; n = 5, WT Cis; n = 5, KO Cis. (G) n = 3, WT CTR; n = 3, KO CTR; n = 3, WT Cis; n = 3, KO Cis. (H) n = 4, WT CTR; n = 4, KO CTR; n = 4, WT Cis; n = 4, KO Cis.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Injection, Staining, Saline, Expressing, Control, Western Blot, Marker

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 9. NAD+ but not NMN alleviates the renal injuries in Ppdpf KO mice with FA treatment. (A) Scheme of the experimental approach. Eight-week-old male mice were intraperitoneally injected with NAD+ (3 mg/g) or NMN (500 mg/kg). FA (250 mg/kg) was injected 3 days later, and mice were then euthanized after 7 days. (B) Serum cre- atinine levels of WT and Ppdpf KO mice with or without FA, NAD+ or NMN treatment. (C) Representative images of H&E- and Sirus red–stained kidney sections from WT and Ppdpf KO mice following FA and NAD injection. Scale bar, 20 μm. (D) Quantifications of tubular injury score and Sirus red staining area in the kidneys of WT and Ppdpf KO mice with or without FA, NMN, or NAD+ administration. (E) Western blots of vimentin in kidneys of WT and Ppdpf KO mice with or without FA and NAD+ treatment. (F) The relative expression of fibrosis markers Fibronectin and Col3a1 in kidneys of WT and Ppdpf KO mice with or without FA, NMN, or NAD+ administration. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 3, WT + FA; n = 3, KO + FA; n = 6, WT + NMN + FA; n = 6, KO + NMN + FA; n = 5, WT + NAD + FA; n = 3, KO + NAD + FA. (D) n = 10 for each group. (E) n = 2 for each group. (F) n = 4, WT; n = 4, KO; n = 3, WT + FA; n = 3, KO + FA; n = 6, WT + NMN + FA; n = 6, KO + NMN + FA; n = 5, WT + NAD + FA; n = 3, KO + NAD + FA.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Injection, Staining, Western Blot, Expressing

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 10. Overexpression of Ppdpf alleviates kidney fibrosis induced by FA administration. (A) Schematic diagram of Ppdpf overexpression in mice. Eight-week-old male mice were in situ injected with AAV9 encoding Ppdpf or scramble. Post–4-week injection, the mice were subjected to FA treatment. (B) The relative expression levels of Ppdpf in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (C) Western blots of Flag and PPDPF in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (D) Western blots of PPDPF in LTL-positive cells from AAV9-Ctrl or AAV9-PPDPF kidneys. (E) Representative images of H&E- and Sirus red–stained kidney sections from AAV9-Ctrl– or AAV9- PPDPF–injected mice following FA administration. Scale bars, 20 μm. (F) Quantifications of tubular injury score and Sirus red staining area from (E). (G) The relative expres- sion levels of fibrosis markers Col3a1, Vimentin, and Fibronectin, as well as injury markers Kim1 and Lcn2 in kidneys of AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. *P < 0.05. (H) Representative images of vimentin, fibronectin, and Col3a1 staining on kidney sections from AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. Scale bars, 50 or 20 μm. (I) NAD+ content of whole kidney lysates from AAV9-Ctrl– or AAV9-PPDPF–injected mice. (J) NMNAT activity of pri- mary cells isolated from AAV9-Ctrl– or AAV9-PPDPF–injected kidneys. P value was calculated by two-tailed t test (B, F, I, and J) and two-way ANOVA (G). P < 0.05 is statisti- cally significant. Data are represented as mean ± SEM. (B) n = 3, AAV9-Ctrl; n = 4, AAV9-PPDPF. (C and D) n = 3 for each group. (F) n = 10 for each group. (G) n = 3, FA-AAV9-Ctrl; n = 4, FA-AAV9-PPDPF. (I) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Over Expression, In Situ, Injection, Expressing, Western Blot, Staining, Activity Assay, Isolation, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 5. PPDPF deficiency inhibits NAD+ synthesis through NMNATs. (A) Representative images of double staining of PPDPF and mitochondrial markers ATP5A1, HSP60, or TOMM20 in HK2 cells. Scale bar, 10 μm. (B) Western blots of subcellular fractions of HK2 cells stained with PPDPF, the mitochondrial antibody TOMM20, and the nuclear antibody LAMIN B1. GAPDH was used as internal control. (C) Schematic diagram of PPDPF engages in the synthesis of NAD+ from NMN. (D) NAD+ content of sham and sgRNA-targeted HK2 cells following PBS or NMN treatment. (E) NAD+ content of primary cells isolated from WT and Ppdpf KO kidney following NMN treatment. (F) NAD/ NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 4 hours and measured with SoNar biosensor. (G) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 12 hours and measured with SoNar biosensor. (H) NMNAT activity of primary cells isolated from WT and Ppdpf KO kidney. A450, absorbance at 450 nm. (I) Representative double staining of PPDPF with NMNAT1, NMNAT2, or NMNAT3 in HK2 cells. (J) Representative images of PLA (red). (K) Quantifications of PLA. Scale bar, 10 μm. P value was calculated by two-tailed t test and one-way analysis of variance (ANOVA). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (D) n = 4 for each group. (E) n = 4, WT; n = 4, KO. (F) n = 230, WT; n = 209, KO. (G) n = 87, WT; n = 119, KO. (H) n = 3, WT; n = 3, KO. (J) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Double Staining, Western Blot, Staining, Control, Isolation, Activity Assay, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 5. PPDPF deficiency inhibits NAD+ synthesis through NMNATs. (A) Representative images of double staining of PPDPF and mitochondrial markers ATP5A1, HSP60, or TOMM20 in HK2 cells. Scale bar, 10 μm. (B) Western blots of subcellular fractions of HK2 cells stained with PPDPF, the mitochondrial antibody TOMM20, and the nuclear antibody LAMIN B1. GAPDH was used as internal control. (C) Schematic diagram of PPDPF engages in the synthesis of NAD+ from NMN. (D) NAD+ content of sham and sgRNA-targeted HK2 cells following PBS or NMN treatment. (E) NAD+ content of primary cells isolated from WT and Ppdpf KO kidney following NMN treatment. (F) NAD/ NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 4 hours and measured with SoNar biosensor. (G) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 12 hours and measured with SoNar biosensor. (H) NMNAT activity of primary cells isolated from WT and Ppdpf KO kidney. A450, absorbance at 450 nm. (I) Representative double staining of PPDPF with NMNAT1, NMNAT2, or NMNAT3 in HK2 cells. (J) Representative images of PLA (red). (K) Quantifications of PLA. Scale bar, 10 μm. P value was calculated by two-tailed t test and one-way analysis of variance (ANOVA). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (D) n = 4 for each group. (E) n = 4, WT; n = 4, KO. (F) n = 230, WT; n = 209, KO. (G) n = 87, WT; n = 119, KO. (H) n = 3, WT; n = 3, KO. (J) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Double Staining, Western Blot, Staining, Control, Isolation, Activity Assay, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 5. PPDPF deficiency inhibits NAD+ synthesis through NMNATs. (A) Representative images of double staining of PPDPF and mitochondrial markers ATP5A1, HSP60, or TOMM20 in HK2 cells. Scale bar, 10 μm. (B) Western blots of subcellular fractions of HK2 cells stained with PPDPF, the mitochondrial antibody TOMM20, and the nuclear antibody LAMIN B1. GAPDH was used as internal control. (C) Schematic diagram of PPDPF engages in the synthesis of NAD+ from NMN. (D) NAD+ content of sham and sgRNA-targeted HK2 cells following PBS or NMN treatment. (E) NAD+ content of primary cells isolated from WT and Ppdpf KO kidney following NMN treatment. (F) NAD/ NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 4 hours and measured with SoNar biosensor. (G) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 12 hours and measured with SoNar biosensor. (H) NMNAT activity of primary cells isolated from WT and Ppdpf KO kidney. A450, absorbance at 450 nm. (I) Representative double staining of PPDPF with NMNAT1, NMNAT2, or NMNAT3 in HK2 cells. (J) Representative images of PLA (red). (K) Quantifications of PLA. Scale bar, 10 μm. P value was calculated by two-tailed t test and one-way analysis of variance (ANOVA). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (D) n = 4 for each group. (E) n = 4, WT; n = 4, KO. (F) n = 230, WT; n = 209, KO. (G) n = 87, WT; n = 119, KO. (H) n = 3, WT; n = 3, KO. (J) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Double Staining, Western Blot, Staining, Control, Isolation, Activity Assay, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 7. Ppdpf deficiency exacerbates renal injury in aged mice. (A) Scheme of the experimental process. Kidneys, serum, and urine were harvested at the age of 12 months. (B) Serum creatinine levels in WT and Ppdpf KO mice at 12 months old. (C) Serum BUN levels in WT and Ppdpf KO mice at 12 months old. (D) Serum cystatin C levels in WT and Ppdpf KO mice at 12 months old. (E) Urine albumin levels in WT and Ppdpf KO mice at 12 months old. (F) Representative images of H&E- and Masson- stained kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bar, 20 μm. (G) Quantification of tubular injury score and Masson staining area from (F). (H) Relative mRNA levels of fibrosis markers Col3a1, Col1a1, and Vimentin in kidneys of WT and Ppdpf KO mice at 12 months old. (I) Western blots and quantifications of vimentin, ACTA2, and KIM1 in kidneys of WT and Ppdpf KO mice at 12 months old. (J) Representative images of vimentin and KIM1 staining in kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bars, 20 μm. (K) NAD+ content of whole kidney lysates of WT and Ppdpf KO mice at 12 months old. P value was calculated by two- tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B and C) n = 4, WT; n = 9, KO. (D) n = 3, WT; n = 5, KO. (E) n = 8, WT; n = 8, KO. (F) n = 6, WT; n = 11, KO. (H) n = 7, WT; n = 13, KO. (H) n = 3, WT; n = 3, KO. (K) n = 4, WT; n = 4, KO.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 8. Ppdpf deficiency exacerbates renal injury in the long-term cisplatin model. (A) Scheme of the experimental approach. Eight-week-old male mice were intra- peritoneally injected with cisplatin (Cis; 7 mg/kg) and euthanized after 28 days. (B) Serum creatinine levels of WT and Ppdpf KO mice with or without cisplatin treatment. (C) Urine albumin levels in WT and Ppdpf KO mice with cisplatin treatment. (D) Representative images of H&E- and Masson-stained kidney sections from WT and Ppdpf KO mice following saline or cisplatin injection. Scale bars, 20 μm. (E) Quantification of tubular injury score and Masson staining area from (D). (F) The relative expression of fibrosis markers Col3a1, Fibronectin, and Vimentin in control or cisplatin-treated WT and Ppdpf KO mice. (G) Western blots of fibrosis markers ACTA2, vimentin, fibronectin, and mitochondrial marker DRP1 in WT and Ppdpf KO mice with or without cisplatin treatment. (H) NAD+ content of whole kidney lysates of WT and Ppdpf KO mice with or without cisplatin treatment. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 4, WT CTR; n = 3, KO CTR; n = 5, WT Cis; n = 5, KO Cis. (C) n = 5, WT Cis; n = 5, KO Cis. (E) n = 3, WT CTR; n = 3, KO CTR; n = 6, WT Cis; n = 6, KO Cis. (F) n = 4, WT CTR; n = 4, KO CTR; n = 5, WT Cis; n = 5, KO Cis. (G) n = 3, WT CTR; n = 3, KO CTR; n = 3, WT Cis; n = 3, KO Cis. (H) n = 4, WT CTR; n = 4, KO CTR; n = 4, WT Cis; n = 4, KO Cis.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Injection, Staining, Saline, Expressing, Control, Western Blot, Marker

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 9. NAD+ but not NMN alleviates the renal injuries in Ppdpf KO mice with FA treatment. (A) Scheme of the experimental approach. Eight-week-old male mice were intraperitoneally injected with NAD+ (3 mg/g) or NMN (500 mg/kg). FA (250 mg/kg) was injected 3 days later, and mice were then euthanized after 7 days. (B) Serum cre- atinine levels of WT and Ppdpf KO mice with or without FA, NAD+ or NMN treatment. (C) Representative images of H&E- and Sirus red–stained kidney sections from WT and Ppdpf KO mice following FA and NAD injection. Scale bar, 20 μm. (D) Quantifications of tubular injury score and Sirus red staining area in the kidneys of WT and Ppdpf KO mice with or without FA, NMN, or NAD+ administration. (E) Western blots of vimentin in kidneys of WT and Ppdpf KO mice with or without FA and NAD+ treatment. (F) The relative expression of fibrosis markers Fibronectin and Col3a1 in kidneys of WT and Ppdpf KO mice with or without FA, NMN, or NAD+ administration. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 3, WT + FA; n = 3, KO + FA; n = 6, WT + NMN + FA; n = 6, KO + NMN + FA; n = 5, WT + NAD + FA; n = 3, KO + NAD + FA. (D) n = 10 for each group. (E) n = 2 for each group. (F) n = 4, WT; n = 4, KO; n = 3, WT + FA; n = 3, KO + FA; n = 6, WT + NMN + FA; n = 6, KO + NMN + FA; n = 5, WT + NAD + FA; n = 3, KO + NAD + FA.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Injection, Staining, Western Blot, Expressing

Journal: Science advances

Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.

doi: 10.1126/sciadv.adr8648

Figure Lengend Snippet: Fig. 10. Overexpression of Ppdpf alleviates kidney fibrosis induced by FA administration. (A) Schematic diagram of Ppdpf overexpression in mice. Eight-week-old male mice were in situ injected with AAV9 encoding Ppdpf or scramble. Post–4-week injection, the mice were subjected to FA treatment. (B) The relative expression levels of Ppdpf in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (C) Western blots of Flag and PPDPF in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (D) Western blots of PPDPF in LTL-positive cells from AAV9-Ctrl or AAV9-PPDPF kidneys. (E) Representative images of H&E- and Sirus red–stained kidney sections from AAV9-Ctrl– or AAV9- PPDPF–injected mice following FA administration. Scale bars, 20 μm. (F) Quantifications of tubular injury score and Sirus red staining area from (E). (G) The relative expres- sion levels of fibrosis markers Col3a1, Vimentin, and Fibronectin, as well as injury markers Kim1 and Lcn2 in kidneys of AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. *P < 0.05. (H) Representative images of vimentin, fibronectin, and Col3a1 staining on kidney sections from AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. Scale bars, 50 or 20 μm. (I) NAD+ content of whole kidney lysates from AAV9-Ctrl– or AAV9-PPDPF–injected mice. (J) NMNAT activity of pri- mary cells isolated from AAV9-Ctrl– or AAV9-PPDPF–injected kidneys. P value was calculated by two-tailed t test (B, F, I, and J) and two-way ANOVA (G). P < 0.05 is statisti- cally significant. Data are represented as mean ± SEM. (B) n = 3, AAV9-Ctrl; n = 4, AAV9-PPDPF. (C and D) n = 3 for each group. (F) n = 10 for each group. (G) n = 3, FA-AAV9-Ctrl; n = 4, FA-AAV9-PPDPF. (I) n = 4 for each group.

Article Snippet: Immunostaining was carried out using the following primary antibodies: PPDPF (Proteintech, #19912- 1- AP), KIM1 (R&D, #AF1817- SP), ATP5A1 (Proteintech, #66037- 1- Ig), HSP60 (Proteintech, #66041- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), NMNAT1 (Abcam, #ab118270; Sigma- Aldrich, #HPA059447), NMNAT2 (Abcam, #ab56980; Sigma- Aldrich, #HPA015708), NMNAT3 (Abcam, #ab71904; Sigma- Aldrich, #HPA039077), NAMPT (Thermo Fisher Scientific, #PA1- 1045), vimentin (Proteintech, #10366- 1- AP), fibronectin (Proteintech, #15613- 1- AP), Col3a1 (Proteintech, #22734- 1- AP), and LTL (Vector, #FL- 1321- 2).

Techniques: Over Expression, In Situ, Injection, Expressing, Western Blot, Staining, Activity Assay, Isolation, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Dimethyl malonate alleviates obstructive nephropathy by enhancing renal metabolism and inhibiting kidney oxidative stress and inflammation

doi: 10.3389/fphar.2025.1530635

Figure Lengend Snippet: DMM inhibited CD4 + T cell infiltration and kidney inflammation. Representative flow cytometry plots of (A) macrophages, (B) monocytes and neutrophils, (C) NK, NKT, and T cells, and (D) B cells in UUO kidneys from mice treated with or without DMM. (E) Quantification of the indicated immune cells in PBS- and DMM-treated UUO kidneys (n = 5). (F) Representative flow cytometry plots and (G) quantification of CD4 + and CD8 + T cells in UUO kidneys from mice treated with or without DMM (n = 5). (H) Immunoblots and (I) quantification of IKK-β, NK-κB p65, and CD38 in UUO kidneys from mice treated with or without DMM (n = 3). (J) qPCR analysis for IL-1β, IL-6, and TNF expression in PBS- or DMM-treated UUO kidneys (n = 6). The results represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following primary antibodies were used: anti-goat KIM1 (R&D, AF1817-SP), anti-αSMA (Proteintech, 67,735-1-lg), anti-Col I (Abcam, ab260043), anti-fibronectin polyclonal antibody (Proteintech, 15613-1-AP), anti-cleaved caspase-3 (CST, 9661S), anti-IKK-β (CST, 2678T), anti-NF-κB p65 (CST, 8242T), anti-CD38 (Santa Cruz, sc-374650), anti-PPARα (Huaxingbio, HX18360), anti-PPARγ polyclonal antibody (Proteintech, 16643-1-AP), anti-β-actin (Huaxingbio, HX 1827), and anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Flow Cytometry, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Dimethyl malonate alleviates obstructive nephropathy by enhancing renal metabolism and inhibiting kidney oxidative stress and inflammation

doi: 10.3389/fphar.2025.1530635

Figure Lengend Snippet: DMM promoted PPAR signaling in UUO kidneys and tubular cells. Immunoblots and quantification of PPARα (A,B) and PPARγ (C,D) in UUO kidneys from mice treated with DMM or succinate (n = 3). (E) qPCR analysis for PPARα and PPARγ expression in TCMK-1 cells treated rhTGF-β or rhTGF-β + DMM (n = 5–6). qPCR analysis for (F) Cpt1a and (G) Acox1 in TCMK-1 cells treated rhTGF-β or rhTGF-β + DMM (n = 6). The results represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, NS no significance.

Article Snippet: The following primary antibodies were used: anti-goat KIM1 (R&D, AF1817-SP), anti-αSMA (Proteintech, 67,735-1-lg), anti-Col I (Abcam, ab260043), anti-fibronectin polyclonal antibody (Proteintech, 15613-1-AP), anti-cleaved caspase-3 (CST, 9661S), anti-IKK-β (CST, 2678T), anti-NF-κB p65 (CST, 8242T), anti-CD38 (Santa Cruz, sc-374650), anti-PPARα (Huaxingbio, HX18360), anti-PPARγ polyclonal antibody (Proteintech, 16643-1-AP), anti-β-actin (Huaxingbio, HX 1827), and anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Dimethyl malonate alleviates obstructive nephropathy by enhancing renal metabolism and inhibiting kidney oxidative stress and inflammation

doi: 10.3389/fphar.2025.1530635

Figure Lengend Snippet: Pharmacological activation of PPAR protected against UUO-induced kidney fibrosis and inflammation. (A) Immunoblots and (B) quantification of FN, Col I, and αSMA in UUO kidneys from mice treated with or without pioglitazone (n = 3). (C) qPCR analysis for FN, Col I, and αSMA in UUO kidneys from mice treated with or without pioglitazone (n = 5). (D) Representative Masson staining and quantification of UUO kidneys from mice treated with or without pioglitazone (n = 5). (E) qPCR analysis for IL-1β and TNF in UUO kidneys from mice treated with or without pioglitazone (n = 5). The results represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following primary antibodies were used: anti-goat KIM1 (R&D, AF1817-SP), anti-αSMA (Proteintech, 67,735-1-lg), anti-Col I (Abcam, ab260043), anti-fibronectin polyclonal antibody (Proteintech, 15613-1-AP), anti-cleaved caspase-3 (CST, 9661S), anti-IKK-β (CST, 2678T), anti-NF-κB p65 (CST, 8242T), anti-CD38 (Santa Cruz, sc-374650), anti-PPARα (Huaxingbio, HX18360), anti-PPARγ polyclonal antibody (Proteintech, 16643-1-AP), anti-β-actin (Huaxingbio, HX 1827), and anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Activation Assay, Western Blot, Staining