Journal: Vascular pharmacology

Article Title: Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability

doi: 10.1016/j.vph.2022.107088

Figure Lengend Snippet: Lack of ASK1 in mice reduces thrombin-induced vascular permeability: (A) Representative images of hind-limb showing vascular leakage of Evan’s blue in WT (left panel) and Ask1−/− mice (right panel) treated locally with thrombin (1 U/ml). (B) Quantification of Evans blue dye extravasation from the hind-limb in WT (n = 3) and Ask1−/− mice (n = 4). Data are represented as mean ± SEM. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: Human thrombin (Enzyme Research Laboratories, South Bend, IN); LPS ( E. coli serotype 0111: B4) (Cell Signaling Technology, Beverly, MA); Phosphoserine 967 ASK1, P-p38, P-JNK1/2, JNK1/2, p38, PMLC2, MLC2 antibodies (Cell Signaling Technology, Beverly, MA); ASK1, Vinculin, VE-cadherin and ZO-1 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); JAM-A antibody (BD Biosciences, San Jose, CA); GS-4997 (ASK1 inhibitor) was a gift from Dr. Anton Simeonov (National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD); Y7632 (ROCK inhibitor; Millipore, Burlington, MA); SB203680 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor; Calbiochem, MA); Vorapaxar (VPX, PAR1 receptor inhibitor) was a kind gift from Dr. Paul Bray (University of Utah Health Science Center, Salt Lake City, UT); BAPTA-AM and Evans blue (Sigma, St. Louis, MO); MnTMPyP (Santa Cruz, CA, USA); S1P (Avanti, INC).

Techniques: Permeability

Journal: Vascular pharmacology

Article Title: Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability

doi: 10.1016/j.vph.2022.107088

Figure Lengend Snippet: Inhibition or deficiency of ASK1 attenuates thrombin-induced endothelial permeability: Representative baseline normalized tracings of CI of unstimulated (US), thrombin (Thr, 1 U/ml), ASK1 inhibitor (ASK1i, 10 μM), and ASK1i + thrombin in (Ai) HUVECs, (Bi) HMVECs, and (Ci) TIME cells. Each line represents the average of two technical replicates. Black arrow represents addition of thrombin (Aii, Bii, and Cii). Quantification of corresponding normalized cell index data (n = 3). (D) Fold change in the amount of FITC dextran passed through the HUVEC monolayer treated with or without thrombin in the presence or absence of ASK1i (n = 4). (E) Representative baseline normalized tracings of cell index of MLECs isolated from WT or Ask1−/− mice, treated with or without thrombin. Black arrow represents addition of thrombin.

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: Human thrombin (Enzyme Research Laboratories, South Bend, IN); LPS ( E. coli serotype 0111: B4) (Cell Signaling Technology, Beverly, MA); Phosphoserine 967 ASK1, P-p38, P-JNK1/2, JNK1/2, p38, PMLC2, MLC2 antibodies (Cell Signaling Technology, Beverly, MA); ASK1, Vinculin, VE-cadherin and ZO-1 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); JAM-A antibody (BD Biosciences, San Jose, CA); GS-4997 (ASK1 inhibitor) was a gift from Dr. Anton Simeonov (National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD); Y7632 (ROCK inhibitor; Millipore, Burlington, MA); SB203680 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor; Calbiochem, MA); Vorapaxar (VPX, PAR1 receptor inhibitor) was a kind gift from Dr. Paul Bray (University of Utah Health Science Center, Salt Lake City, UT); BAPTA-AM and Evans blue (Sigma, St. Louis, MO); MnTMPyP (Santa Cruz, CA, USA); S1P (Avanti, INC).

Techniques: Inhibition, Permeability, Isolation

Journal: Vascular pharmacology

Article Title: Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability

doi: 10.1016/j.vph.2022.107088

Figure Lengend Snippet: Thrombin induces permeability via ASK1/JNK, and not ASK1/p38-dependent pathway in HUVECs. (Ai) Representative western blot of HUVEC lysate treated with thrombin (1 U/ml) for various time points (1, 2, 5, 10, and 15 min) and probed with antibodies specific for phospho-ASK1 (Ser-967), P-p38 (Thr180/Tyr182), P-JNK1/2(Thr183/Tyr185). Blots were reprobed with anti-ASK1, anti-p38, and anti-JNK1/2 antibodies to ensure equal protein loading. (Aii) Quantification of band densities from (A) expressed as ratio of phospho-protein to total protein. (n = 3). (Bi) Representative baseline normalized tracings of cell index of HUVECs unstimulated (US), or pretreated with p38i, JNKi, or both prior to stimulation with thrombin (1 U/ml). (Bii) Quantification of cell index data from (Bi) (n = 3). *p < 0.5, **p < 0.001, ***p < 0.001.

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: Human thrombin (Enzyme Research Laboratories, South Bend, IN); LPS ( E. coli serotype 0111: B4) (Cell Signaling Technology, Beverly, MA); Phosphoserine 967 ASK1, P-p38, P-JNK1/2, JNK1/2, p38, PMLC2, MLC2 antibodies (Cell Signaling Technology, Beverly, MA); ASK1, Vinculin, VE-cadherin and ZO-1 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); JAM-A antibody (BD Biosciences, San Jose, CA); GS-4997 (ASK1 inhibitor) was a gift from Dr. Anton Simeonov (National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD); Y7632 (ROCK inhibitor; Millipore, Burlington, MA); SB203680 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor; Calbiochem, MA); Vorapaxar (VPX, PAR1 receptor inhibitor) was a kind gift from Dr. Paul Bray (University of Utah Health Science Center, Salt Lake City, UT); BAPTA-AM and Evans blue (Sigma, St. Louis, MO); MnTMPyP (Santa Cruz, CA, USA); S1P (Avanti, INC).

Techniques: Permeability, Western Blot

Journal: Vascular pharmacology

Article Title: Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability

doi: 10.1016/j.vph.2022.107088

Figure Lengend Snippet: Thrombin-induced endothelial permeability is dependent on ASK1, Ca2+, and ROCK, but not ROS: (Ai) Representative baseline normalized tracings of cell index of HUVECs unstimulated (US) or stimulated with PAR1-activating peptide, (PAR1-AP, 50 μM), in the presence or absence of ASK1i, 10 μM. (Aii) Quantification of CI data from (Ai) (n = 3). (Bi) Representative baseline normalized tracings of cell index of HUVECs unstimulated (US), pretreated with MnTMPyP (10 μM), BAPTA-AM (10 μM) and stimulated with thrombin (1 U/ ml). Black arrow represents addition of thrombin. (Bii) Quantification of cell index data from (Bi) (n = 3). (Ci) Representative baseline normalized tracings of cell index of HUVECs unstimulated (US), pretreated with VPX (100 nM), ROCKi (10 μM), and stimulated with thrombin (1 U/ml). (Cii) Quantification of normalized cell index data from (Ci) (n = 3). Each line of tracing represents the average of two technical replicates. *p < 0.5, **p < 0.001, ***p < 0.001.

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: Human thrombin (Enzyme Research Laboratories, South Bend, IN); LPS ( E. coli serotype 0111: B4) (Cell Signaling Technology, Beverly, MA); Phosphoserine 967 ASK1, P-p38, P-JNK1/2, JNK1/2, p38, PMLC2, MLC2 antibodies (Cell Signaling Technology, Beverly, MA); ASK1, Vinculin, VE-cadherin and ZO-1 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); JAM-A antibody (BD Biosciences, San Jose, CA); GS-4997 (ASK1 inhibitor) was a gift from Dr. Anton Simeonov (National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD); Y7632 (ROCK inhibitor; Millipore, Burlington, MA); SB203680 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor; Calbiochem, MA); Vorapaxar (VPX, PAR1 receptor inhibitor) was a kind gift from Dr. Paul Bray (University of Utah Health Science Center, Salt Lake City, UT); BAPTA-AM and Evans blue (Sigma, St. Louis, MO); MnTMPyP (Santa Cruz, CA, USA); S1P (Avanti, INC).

Techniques: Permeability

Journal: Vascular pharmacology

Article Title: Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability

doi: 10.1016/j.vph.2022.107088

Figure Lengend Snippet: Inhibition of ASK1 attenuates thrombin-induced disorganization of junctional molecules, para-cellular gap formation in HUVEC monolayer: Immunofluorescence images of serum-starved confluent HUVEC monolayer pretreated with vehicle or ASK1i (10 μM) prior to stimulation with thrombin (2 U/ml) for 20 min. (A) HUVECs were stained (red) with VE-cadherin (upper row), JAM-A (lower row), and counter stained with DAPI (blue). White arrows show loss of junction molecules at the cell-cell contact upon treatment with thrombin (20× images with scale = 20 mm). (B) Following treatment, cells were fixed as usual and stained for DAPI and F-actin (green) at different conditions; unstimulated (US), thrombin (1 U/ml), ASK1 inhibitor (10 μM), and ASK1i + thrombin (60× images with scale = 50 mm). Dotted lines represent boundary for gap area present in between the cells. (C) Quantification of percent area of cell coverage from (n = 3) individual experiments. (D) Representative western-blot image of VE-cadherin cleavage in the presence of thrombin, C-terminal fragment (CTF) of VE-cadherin, and β-actin as loading control. (E) Quantification of band density from (D) expressed as ratio of C-terminal fragment to full length VE-cadherin. (n = 3). *p < 0.5, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: Human thrombin (Enzyme Research Laboratories, South Bend, IN); LPS ( E. coli serotype 0111: B4) (Cell Signaling Technology, Beverly, MA); Phosphoserine 967 ASK1, P-p38, P-JNK1/2, JNK1/2, p38, PMLC2, MLC2 antibodies (Cell Signaling Technology, Beverly, MA); ASK1, Vinculin, VE-cadherin and ZO-1 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); JAM-A antibody (BD Biosciences, San Jose, CA); GS-4997 (ASK1 inhibitor) was a gift from Dr. Anton Simeonov (National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD); Y7632 (ROCK inhibitor; Millipore, Burlington, MA); SB203680 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor; Calbiochem, MA); Vorapaxar (VPX, PAR1 receptor inhibitor) was a kind gift from Dr. Paul Bray (University of Utah Health Science Center, Salt Lake City, UT); BAPTA-AM and Evans blue (Sigma, St. Louis, MO); MnTMPyP (Santa Cruz, CA, USA); S1P (Avanti, INC).

Techniques: Inhibition, Immunofluorescence, Staining, Western Blot, Control

Journal: Vascular pharmacology

Article Title: Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability

doi: 10.1016/j.vph.2022.107088

Figure Lengend Snippet: Inhibition of ASK1 does not affect thrombin-induced cytoskeletal remodeling or phosphorylation of MLC. (A) Immunofluorescence images of serum starved confluent HUVEC monolayer unstimulated (US) or pretreated with 10 μM ASK1i for 30 min prior to stimulation with thrombin (2 U/ml) for 20 min or S1P (5 μM) for 10 min. Cells were stained with vinculin (red), phalloidin (green), and DAPI (blue). Images represents n = 3 experiments. (63× images with scale = 50 μm). (B) Representative western blot showing effect of ASK1i or ROCKi on thrombin-induced MLC2 phosphorylation. (C) Quantification of band density from (B) expressed as ratio of P-MLC2 to total MLC2. (n = 3). **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: Human thrombin (Enzyme Research Laboratories, South Bend, IN); LPS ( E. coli serotype 0111: B4) (Cell Signaling Technology, Beverly, MA); Phosphoserine 967 ASK1, P-p38, P-JNK1/2, JNK1/2, p38, PMLC2, MLC2 antibodies (Cell Signaling Technology, Beverly, MA); ASK1, Vinculin, VE-cadherin and ZO-1 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); JAM-A antibody (BD Biosciences, San Jose, CA); GS-4997 (ASK1 inhibitor) was a gift from Dr. Anton Simeonov (National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD); Y7632 (ROCK inhibitor; Millipore, Burlington, MA); SB203680 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor; Calbiochem, MA); Vorapaxar (VPX, PAR1 receptor inhibitor) was a kind gift from Dr. Paul Bray (University of Utah Health Science Center, Salt Lake City, UT); BAPTA-AM and Evans blue (Sigma, St. Louis, MO); MnTMPyP (Santa Cruz, CA, USA); S1P (Avanti, INC).

Techniques: Inhibition, Phospho-proteomics, Immunofluorescence, Staining, Western Blot

Journal: Biology Direct

Article Title: TIMM8B promotes oxidative phosphorylation and glycolysis by inhibiting the mtROS/ASK1/JNK signaling pathway in ovarian cancer

doi: 10.1186/s13062-025-00663-6

Figure Lengend Snippet: TIMM8B inhibited the activation of the mtROS/ASK1/JNK pathway. A The effects of TIMM8B on the ASK1/JNK signaling pathway were determined by Western blotting assays. B The mtROS scavenger Mito-TEMPO (MT), which is a mitochondria-targeted superoxide dismutase mimetic, reduced the effects of TIMM8B on the ASK1/JNK signaling pathway, as determined by Western blotting assays; ** P < 0.01, compared to the sh-NC group; ## P < 0.01, compared to the Lv group; $$ P < 0.01, compared to the sh-TIMM8B group

Article Snippet: The membranes were incubated with the following primary antibodies: TIMM8B (1:1000, abs152377, Absin), cyclin E1 (1:2000, AF0144, Affinity), p27 (1:1000, 3686S, Cell Signaling Technology), cyclin D1 (1:3000, ab134175, Abcam), p21 (1:1000, 14472S, Cell Signaling Technology), E-cadherin (1:1000, 14472S, Cell Signaling Technology), vimentin (1:1000, ab8978, Abcam), Snail (1:1000, ab216347, Abcam), N-cadherin (1:1000, 13116S, Cell Signaling Technology), BCL-XL (1:1000, AF6414, Affinity), BCL-2 (1:1000, AF6139, Affinity), BAX (1:2000, 29,552–1-AP, Proteintech), cleaved-Caspase 3 (1:2000, AF0722, Affinity), cleaved-Caspase 7 (1:2000, AF4023, Affinity), NDUFS1 (Complex I, 1:1000, DF7041, Affinity), SDHB (Complex II, 1:1000, DF12732, Affinity), CYTB polyclonal (Complex III, 1:2000, 55,090–1-AP, Proteintech); Complex IV (1:1000, 4850, Cell Signaling), ATP5A (Complex V, 1:1000, ab14748, Abcam), ASK1 (1:1000, 8662S, Cell Signaling Technology), p-JNK (1:1000, AF3318, Affinity), JNK (1:1000, AF6318, Affinity), and GAPDH (1:5000, AB0037, Abways) at 4 °C overnight.

Techniques: Activation Assay, Western Blot

Journal: Biology Direct

Article Title: TIMM8B promotes oxidative phosphorylation and glycolysis by inhibiting the mtROS/ASK1/JNK signaling pathway in ovarian cancer

doi: 10.1186/s13062-025-00663-6

Figure Lengend Snippet: TIMM8B inhibited mtROS/ASK1/JNK to regulate the behavior and energy metabolism in the SKOV3 and OVCAR3 cell lines. A A colony formation assay was performed in TIMM8B-overexpressing cells after treatment with AM (JNK agonist, 1 μg/mL). B Flow cytometry was conducted to determine cell cycle progression in TIMM8B-overexpressing cells after treatment with AM. C Western blotting assays were performed to measure the levels of cyclin proteins in TIMM8B-overexpressing cells after treatment with AM. D and E Cell invasion and migration were determined by conducting a Transwell assay in TIMM8B-overexpressing cells after treatment with AM. F Western blotting analysis was performed to measure the levels of EMT-related proteins (N-cadherin, vimentin, snail, and E-cadherin) in TIMM8B-overexpressing cells after treatment with AM. G Cell apoptosis was detected by flow cytometry in TIMM8B-overexpressing cells after treatment with AM. H and I Western blotting analysis was performed to measure the levels of apoptosis-related proteins (BCL 2, BCL-XL, BAX, BAK, cleaved caspase 3, and cleaved caspase 7) in TIMM8B-overexpressing cells after treatment with AM; ** P < 0.01, compared to the sh-NC group; ## P < 0.01, compared to the TIMM8B group

Article Snippet: The membranes were incubated with the following primary antibodies: TIMM8B (1:1000, abs152377, Absin), cyclin E1 (1:2000, AF0144, Affinity), p27 (1:1000, 3686S, Cell Signaling Technology), cyclin D1 (1:3000, ab134175, Abcam), p21 (1:1000, 14472S, Cell Signaling Technology), E-cadherin (1:1000, 14472S, Cell Signaling Technology), vimentin (1:1000, ab8978, Abcam), Snail (1:1000, ab216347, Abcam), N-cadherin (1:1000, 13116S, Cell Signaling Technology), BCL-XL (1:1000, AF6414, Affinity), BCL-2 (1:1000, AF6139, Affinity), BAX (1:2000, 29,552–1-AP, Proteintech), cleaved-Caspase 3 (1:2000, AF0722, Affinity), cleaved-Caspase 7 (1:2000, AF4023, Affinity), NDUFS1 (Complex I, 1:1000, DF7041, Affinity), SDHB (Complex II, 1:1000, DF12732, Affinity), CYTB polyclonal (Complex III, 1:2000, 55,090–1-AP, Proteintech); Complex IV (1:1000, 4850, Cell Signaling), ATP5A (Complex V, 1:1000, ab14748, Abcam), ASK1 (1:1000, 8662S, Cell Signaling Technology), p-JNK (1:1000, AF3318, Affinity), JNK (1:1000, AF6318, Affinity), and GAPDH (1:5000, AB0037, Abways) at 4 °C overnight.

Techniques: Colony Assay, Flow Cytometry, Western Blot, Migration, Transwell Assay

Journal: Biology Direct

Article Title: TIMM8B promotes oxidative phosphorylation and glycolysis by inhibiting the mtROS/ASK1/JNK signaling pathway in ovarian cancer

doi: 10.1186/s13062-025-00663-6

Figure Lengend Snippet: TIMM8B inhibited mtROS/ASK1/JNK to regulate energy metabolism in the SKOV3 and OVCAR3 cell lines. A Hexokinase activity was measured in TIMM8B-overexpressing cells after AM treatment. B Lactic acid production was evaluated in TIMM8B-overexpressing cells after AM treatment. C ATP concentrations were assessed in TIMM8B-overexpressing cells after AM treatment. D and E Western blotting analysis was performed to measure the levels of mitochondrial complex subunits; ** P < 0.01, compared to the sh-NC group; ## P < 0.01, compared to the TIMM8B group

Article Snippet: The membranes were incubated with the following primary antibodies: TIMM8B (1:1000, abs152377, Absin), cyclin E1 (1:2000, AF0144, Affinity), p27 (1:1000, 3686S, Cell Signaling Technology), cyclin D1 (1:3000, ab134175, Abcam), p21 (1:1000, 14472S, Cell Signaling Technology), E-cadherin (1:1000, 14472S, Cell Signaling Technology), vimentin (1:1000, ab8978, Abcam), Snail (1:1000, ab216347, Abcam), N-cadherin (1:1000, 13116S, Cell Signaling Technology), BCL-XL (1:1000, AF6414, Affinity), BCL-2 (1:1000, AF6139, Affinity), BAX (1:2000, 29,552–1-AP, Proteintech), cleaved-Caspase 3 (1:2000, AF0722, Affinity), cleaved-Caspase 7 (1:2000, AF4023, Affinity), NDUFS1 (Complex I, 1:1000, DF7041, Affinity), SDHB (Complex II, 1:1000, DF12732, Affinity), CYTB polyclonal (Complex III, 1:2000, 55,090–1-AP, Proteintech); Complex IV (1:1000, 4850, Cell Signaling), ATP5A (Complex V, 1:1000, ab14748, Abcam), ASK1 (1:1000, 8662S, Cell Signaling Technology), p-JNK (1:1000, AF3318, Affinity), JNK (1:1000, AF6318, Affinity), and GAPDH (1:5000, AB0037, Abways) at 4 °C overnight.

Techniques: Activity Assay, Western Blot

Journal: Nutrition Research and Practice

Article Title: Involvement of miR-Let7A in inflammatory response and cell survival/apoptosis regulated by resveratrol in THP-1 macrophage

doi: 10.4162/nrp.2016.10.4.377

Figure Lengend Snippet: Immunofluorescence staining was performed to check the activation of ASK1 (p-ASK1). Confocal microscopy analysis was performed to visualize the activation of ASK1 (p-ASK1). THP-1 cells were pretreated with resveratrol or/and Let7A mimic separately or following stimulation with LPS. p-ASK1 is represented by green staining, nuclear DNA is indicated by DAPI staining (blue color), and the combined images are presented; Normal: non-treated cells, Let7A (miR-Let7A mimic): Let7A overexpression, Resv: resveratrol (25 µM), LPS: lipopolysaccharide (1 µg/ml)

Article Snippet: After blocking with 5% BSA prepared in TBS/Tween (20 nM Tris (pH 7.2), 150 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature, immunoblots were incubated overnight at 4°C with primary antibodies specifically for detection of caspase-3 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 (1:1000, Santa Cruz Biotechnology), ASK1 (1:1000; Santa Cruz Biotechnology), phosphorylated ASK1 (p-ASK1; 1:1000; Santa Cruz Biotechnology), or β-actin (1:1000; Cell Signaling, Danvers, MA, USA).

Techniques: Immunofluorescence, Staining, Activation Assay, Confocal Microscopy, Over Expression

Journal: Journal of Biological Chemistry

Article Title: Requirement of Reactive Oxygen Species-dependent Activation of ASK1-p38 MAPK Pathway for Extracellular ATP-induced Apoptosis in Macrophage

doi: 10.1074/jbc.m708402200

Figure Lengend Snippet: FIGURE 5. ASK1 is required for ATP-induced apoptosis in macrophages. A, immunoblot analysis of phospho- ASK1inlysatesofASK1/ andASK1/ mouseSDMsstimulatedwith1mMATPor0.3mMH2O2 for the indicated periods. B, immunoblot analysis of phospho-p38 and p38 in lysates of ASK1/ and ASK1/ SDMs stim- ulated with 1 mM ATP or 0.3 mM H2O2 for the indicated periods (upper panel). Relative activities of p38 are shown in the graph (lower panel). C and D, ASK1/ and ASK1/ SDMs were treated with 3 mM ATP or 0.3 mM H2O2. After 6 h, the cells were lysed and caspase-3 protease activity (C) and the quantity of cytoplasmic histone- associated DNA fragments (D) were measured as described under “Materials and Methods.” The data are the meansS.E.(n3).E,theexpressionlevelsofP2X7receptorand-actin(asaninertialcontrol)inASK1/and ASK1/ SDMs were analyzed by immunoblotting. F, ATP-mediated ROS production was measured using HPF. RelativeROSproductionat2and5minafterexposureof2mMATP(normalizedwithbasalROSproduction)was measured in ASK1/ and ASK1/ SDMs. The data are the means S.E. (n 3).

Article Snippet: The membranes were probed with antibodies to phospho-ASK1 (24), phospho-p38 (Cell Signaling Technology), P2X7 (Y-14), p38 (C-20), NOX2 (C-15) (Santa Cruz), -actin, and FLAG (M2) (Sigma).

Techniques: Western Blot, Activity Assay