dgk α  (Proteintech)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: CAM12203 directly binds to DGK ζ . (A) Structure of the biotinylated probe. (B) RAW264.7 cells (pretreated with or without the probe for 2 h) were stimulated with LPS for 4 h. The mRNA level of Il1b was detected by Q-PCR. (C) BMDMs (pretreated with or without the probe for 2 h) were stimulated with LPS (4 h) + ATP (another 45 min). The IL-1 β concentration in culture medium (CM) was examined by HTRF. (D) Overall scheme of the experiments to identify the targets of CAM12203 (created with Biorender.com ). (E) The list of gene names of CAM12203 -binding proteins. (F) RAW264.7 lysate was pretreated with or without CAM12203 for 2 h (4 °C), followed by incubation with the probe for another 2 h (4 °C). The level of DGK ζ pulled down was determined by Western blotting. (G) RAW264.7 lysate was treated with or without CAM12203 , and the protein stability of DGK ζ resistant to pronase E was determined by DARTS. (H) RAW264.7 lysate was treated with or without CAM12203 , and the thermostability of DGK ζ was determined by CETSA. (I) DGK ζ protein was treated with CAM12203 , and the binding affinity was determined by SPR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Concentration Assay, Binding Assay, Incubation, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ plays a critical role in macrophage-mediated inflammation and liver failure. (A) The protein level of DGK ζ in LPS-stimulated RAW264.7 cells was determined by Western blotting. (B–D) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS. The level of PA (B) and IL-1 β expression (C, D) were measured by a commercial kit, Q-PCR, and Western blotting, respectively. (E) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The IL-1 β expression was detected by Q-PCR and Western blotting. (F–H) BMDMs from wild-type (WT) and Dgkz −/− mice were stimulated with LPS + ATP. The culture medium (CM) was collected for examination of IL-1 β concentration (F) by HTRF, and added to the primary hepatocytes along with D-GalN. The levels of cleaved-PARP (c-PARP) and cleaved-Caspase 3 (c-Caspase 3) (G), and the activity of Caspase 3 (scar bar: 100 μm) (H) were determined. (I, J) ALF was induced in mice by LPS + D-GalN. After 1.5, 3 and 4 h, the protein level of DGK ζ in the liver was determined by Western blotting (I). After 6 h, the protein levels of DGK ζ in F4/80 + and Ly6G + cells sorted from the liver were determined by Western blotting (J). (K) Hepatic DGK ζ + CD68 + cells in Healthy Controls ( n = 6) and ACLF patients ( n = 5) were marked (scar bar: 50 μm) and counted. (L–N) WT and Dgkz −/− mice were administered with LPS + D-GalN for 6 h. The liver index (L), the levels of ALT, AST and LDH in plasma (M), the histological alterations of the liver (scar bar: 50 μm) (N), the frequency of MoMFs in liver (O), the mRNA level of Il1b in liver (P), and the IL-1 β concentration in plasma (Q) were evaluated. Data are displayed as mean ± SEM (For A–H, n = 3; For I, J, I–N, n = 6). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Western Blot, Expressing, Transfection, Concentration Assay, Activity Assay, Clinical Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ –STAT3 axis drives IL-1 β production in macrophages. (A–C) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for the indicated time. The levels of pp65, pSTAT3 Ser727 , and pSTAT3 Tyr705 , as well as the nuclear translocation of STAT3 were determined by Western blotting. (D) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (E) Regulators of the JAK2–STAT3 pathway (created with Biorender.com ). (F, G) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for 4 h. The levels of pJAK2 (F), SOCS1, and SOCS3 (G) were determined by Western blotting. (H) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without TPI-1 (3 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 were determined by Western blotting. (I) RAW264.7 cells (with or without U73122 (10 μmol/L), 2-APB (50 μmol/L), and BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 as well as IL-1 β expression were determined by Western blotting and Q-PCR. (J, K) RAW264.7 cells (with or without BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The binding between Pyk2 and JAK2 (J) was examined by immunoprecipitation, and the levels of pPyk2 and pJAK2 (K) were determined by Western blotting. (L, M) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS as well as PA for 4 h. The concentration of Ca 2+ (L) was measured by Fluo-4 AM (scar bar: 50 μm). The levels of pPyk2, pJAK2, and pSTAT3 Tyr705 as well as IL-1 β expression (M) were determined by Western blotting and Q-PCR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Translocation Assay, Western Blot, Transfection, Expressing, Binding Assay, Immunoprecipitation, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: The anti-inflammatory effect of CAM12203 relies on DGK ζ . (A) Overall scheme of the examination of DGK ζ activity (created with Biorender.com ). (B) The effects of BAY 2965501 (BAY) and CAM12203 on DGK ζ activity were measured. (C) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 0.25 h. The level of PA was measured by a commercial kit. (D–F) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The levels of pPyk2, pJAK2 (D) and pSTAT3 Tyr705 (E), and the cytosol as well as nuclear levels of STAT3 (F) were determined by Western blotting. (G) PMA-differentiated THP-1 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (H, I) Mice were administered by CAM12203 (30 mg/kg), and ALF was induced by LPS + D-GalN. After 6 h, the levels of pPyk2, pJAK2 (H), and pSTAT3 Tyr705 (I) in F4/80 + cells sorted from the liver were determined by Western blotting. (J–L) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without CAM12203 /BAY pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 and IL-1 β expression were determined by Western blotting and Q-PCR. (M) RAW264.7 cells were transfected with Control or DGK ζ -OE plasmid, and treated with or without CAM12203 /BAY for 2 h, followed by LPS simulation for 4 h. The levels of pSTAT3 Tyr705 and pro-IL-1 β were determined by Western blotting. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group; ns, not significant.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Control, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: CAM12203 directly binds to DGK ζ . (A) Structure of the biotinylated probe. (B) RAW264.7 cells (pretreated with or without the probe for 2 h) were stimulated with LPS for 4 h. The mRNA level of Il1b was detected by Q-PCR. (C) BMDMs (pretreated with or without the probe for 2 h) were stimulated with LPS (4 h) + ATP (another 45 min). The IL-1 β concentration in culture medium (CM) was examined by HTRF. (D) Overall scheme of the experiments to identify the targets of CAM12203 (created with Biorender.com ). (E) The list of gene names of CAM12203 -binding proteins. (F) RAW264.7 lysate was pretreated with or without CAM12203 for 2 h (4 °C), followed by incubation with the probe for another 2 h (4 °C). The level of DGK ζ pulled down was determined by Western blotting. (G) RAW264.7 lysate was treated with or without CAM12203 , and the protein stability of DGK ζ resistant to pronase E was determined by DARTS. (H) RAW264.7 lysate was treated with or without CAM12203 , and the thermostability of DGK ζ was determined by CETSA. (I) DGK ζ protein was treated with CAM12203 , and the binding affinity was determined by SPR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Concentration Assay, Binding Assay, Incubation, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ plays a critical role in macrophage-mediated inflammation and liver failure. (A) The protein level of DGK ζ in LPS-stimulated RAW264.7 cells was determined by Western blotting. (B–D) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS. The level of PA (B) and IL-1 β expression (C, D) were measured by a commercial kit, Q-PCR, and Western blotting, respectively. (E) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The IL-1 β expression was detected by Q-PCR and Western blotting. (F–H) BMDMs from wild-type (WT) and Dgkz −/− mice were stimulated with LPS + ATP. The culture medium (CM) was collected for examination of IL-1 β concentration (F) by HTRF, and added to the primary hepatocytes along with D-GalN. The levels of cleaved-PARP (c-PARP) and cleaved-Caspase 3 (c-Caspase 3) (G), and the activity of Caspase 3 (scar bar: 100 μm) (H) were determined. (I, J) ALF was induced in mice by LPS + D-GalN. After 1.5, 3 and 4 h, the protein level of DGK ζ in the liver was determined by Western blotting (I). After 6 h, the protein levels of DGK ζ in F4/80 + and Ly6G + cells sorted from the liver were determined by Western blotting (J). (K) Hepatic DGK ζ + CD68 + cells in Healthy Controls ( n = 6) and ACLF patients ( n = 5) were marked (scar bar: 50 μm) and counted. (L–N) WT and Dgkz −/− mice were administered with LPS + D-GalN for 6 h. The liver index (L), the levels of ALT, AST and LDH in plasma (M), the histological alterations of the liver (scar bar: 50 μm) (N), the frequency of MoMFs in liver (O), the mRNA level of Il1b in liver (P), and the IL-1 β concentration in plasma (Q) were evaluated. Data are displayed as mean ± SEM (For A–H, n = 3; For I, J, I–N, n = 6). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Western Blot, Expressing, Transfection, Concentration Assay, Activity Assay, Clinical Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ –STAT3 axis drives IL-1 β production in macrophages. (A–C) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for the indicated time. The levels of pp65, pSTAT3 Ser727 , and pSTAT3 Tyr705 , as well as the nuclear translocation of STAT3 were determined by Western blotting. (D) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (E) Regulators of the JAK2–STAT3 pathway (created with Biorender.com ). (F, G) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for 4 h. The levels of pJAK2 (F), SOCS1, and SOCS3 (G) were determined by Western blotting. (H) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without TPI-1 (3 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 were determined by Western blotting. (I) RAW264.7 cells (with or without U73122 (10 μmol/L), 2-APB (50 μmol/L), and BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 as well as IL-1 β expression were determined by Western blotting and Q-PCR. (J, K) RAW264.7 cells (with or without BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The binding between Pyk2 and JAK2 (J) was examined by immunoprecipitation, and the levels of pPyk2 and pJAK2 (K) were determined by Western blotting. (L, M) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS as well as PA for 4 h. The concentration of Ca 2+ (L) was measured by Fluo-4 AM (scar bar: 50 μm). The levels of pPyk2, pJAK2, and pSTAT3 Tyr705 as well as IL-1 β expression (M) were determined by Western blotting and Q-PCR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Translocation Assay, Western Blot, Transfection, Expressing, Binding Assay, Immunoprecipitation, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: The anti-inflammatory effect of CAM12203 relies on DGK ζ . (A) Overall scheme of the examination of DGK ζ activity (created with Biorender.com ). (B) The effects of BAY 2965501 (BAY) and CAM12203 on DGK ζ activity were measured. (C) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 0.25 h. The level of PA was measured by a commercial kit. (D–F) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The levels of pPyk2, pJAK2 (D) and pSTAT3 Tyr705 (E), and the cytosol as well as nuclear levels of STAT3 (F) were determined by Western blotting. (G) PMA-differentiated THP-1 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (H, I) Mice were administered by CAM12203 (30 mg/kg), and ALF was induced by LPS + D-GalN. After 6 h, the levels of pPyk2, pJAK2 (H), and pSTAT3 Tyr705 (I) in F4/80 + cells sorted from the liver were determined by Western blotting. (J–L) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without CAM12203 /BAY pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 and IL-1 β expression were determined by Western blotting and Q-PCR. (M) RAW264.7 cells were transfected with Control or DGK ζ -OE plasmid, and treated with or without CAM12203 /BAY for 2 h, followed by LPS simulation for 4 h. The levels of pSTAT3 Tyr705 and pro-IL-1 β were determined by Western blotting. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group; ns, not significant.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Control, Plasmid Preparation