n 240  (Alomone Labs)

Journal: Journal of Neurochemistry

Article Title: BH4 Oxidation‐Derived H 2 O 2 Activates ERK1 /2 Signaling via B‐Raf in Rat Dorsal Root Ganglion Neurons

doi: 10.1111/jnc.70271

Figure Lengend Snippet: BH4 exposure increases pERK1/2 levels in rat dorsal root ganglion neurons in a time‐ and dose‐dependent manner. (A) Schematic workflow of performed experiments to analyze ERK1/2 activation via determining pERK1/2 levels in ex vivo cultured rat dorsal root ganglion (DRG) neurons by high‐content imaging microscopy and script‐based processing of single‐cell data. (B) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to increasing concentrations of BH4 or PBS for up to 120 min. Statistical significance tested between each concentration and control by two‐way ANOVA with Bonferroni's test (* p < 0.05; 25 μM: F (1, 36) = 9.481, p = 0.0040; 50 μM: F (1, 36) = 51.43, p < 0.0001; 100 μM: F (1, 36) = 161.8, p < 0.0001). Data presented as mean ± SD. (C) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to 100 ng/mL neural growth factor (NGF), 100 ng/mL Oncostatin M (OSM), 100 μM BH4, or PBS as control. Statistical significance tested by one‐way ANOVA with Bonferroni multiple comparison testing between each condition and PBS (* p < 0.05). F (3, 12) = 3.028, p = 0.0712. Data presented as individual data points representing biological replicates and mean ± SD. (D) Cell density plots showing single cell data of relative pERK1/2 intensity versus relative RIIβ intensity (whole‐cell average set to 1) (upper panel), or versus soma size (lower panel) from neurons shown in (C). (B–D) Experiments have been performed with independent DRG preparations from n = 4 animals.

Article Snippet: For stimulations, BH4 (Sigma, cat. no. T4425), NGF (Alomone labs, cat. no. N‐240), OSM (Peprotech, cat. no. 400‐36), ascorbic acid (Sigma, cat. no. A4403), phenyl‐alpha‐tert‐butyl nitrone (PBN) (Enzo, cat. no. ALX‐430‐082‐G001), BH2 (MCE MedChemExpress, cat. no. HY‐W008646), H 2 O 2 (Applichem, cat. no. A2726,1000), catalase (Sigma, cat. no. C30‐100MG), superoxide dismutase (Sigma, cat. no. S9697‐15KU), 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol) (Selleckchem, cat. no. S2910), ML171 (also 2‐APT) (Tocris, cat. no. 4653), AITC (Sigma, cat. no. 377430), capsaicin (Sigma, cat. no. M2028), L‐NAME (Cayman, 51298‐62‐5, cat. no. 80210), L‐NNA (Tocris, cat. no. 0664), L‐NMMA (Tocris, cat. no. 0771), ruthenium red (Abcam, cat. no. ab120264), 2‐Aminoethyl diphenylborinate (2‐APB) (Calbiochem, cat. no. 100065), EGTA (Sigma, cat. no. E4378), BAPTA‐AM (Abcam, cat. no. ab120503), Trametinib (ApexBio, cat. no. A3018), U0126 (Calbiochem, cat. no. 662005), Lifirafenib (Chemietek, cat. no. CT‐BGB283), SB590855 (Axonmedchem, cat. no. Axon 2504), GDC0879 (Selleckchem, cat. no. S110), GW5074 (Selleckchem, cat. no. S2872), ZM336372 (Sigma, cat. no. SML0236), Dabrafenib (Selleckchem, cat. no. S2807), Sorafenib (Tocris, cat. no. 6814), Salirasib (Sigma, cat. no. SML1166), Dasatinib (Sigma, cat. no. CDS023389), Saracatinib (Selleckchem, cat. no. S1006), Src inhibitor 1 (Sigma, cat. no. S2075), Edelfosine (cat. no. 3022), U73122 (Tocris, cat. no. 1268), GO6983 (Peprotech, cat. no. 1331975), Thapsigargin (Calbiochem, cat. no. 586005), Ryanodine (Calbiochem, cat. no. 559276), Dantrolene (Tocris, cat. no. 507), JTV‐519 (Sigma, cat. no. SML0549), Heparin (Tocris, cat. no. 2812), Flufenamic acid (Tocris, cat. no. 4522), N‐(p‐amylcinnamoyl)anthranilic acid (ACAA) (Abcam, cat. no. ab141555), M‐8B (Tocris, cat. no. 5324), STO‐609 (Tocris, cat. no. 1551), KN‐93 (Calbiochem, cat. no. 422708), and Autocamtide‐2‐Related Inhibitory Peptide (AIP) (Calbiochem, cat. no. 189485) were used.

Techniques: Activation Assay, Ex Vivo, Cell Culture, Imaging, Microscopy, Concentration Assay, Control, Comparison

Journal: Journal of Neurochemistry

Article Title: BH4 Oxidation‐Derived H 2 O 2 Activates ERK1 /2 Signaling via B‐Raf in Rat Dorsal Root Ganglion Neurons

doi: 10.1111/jnc.70271

Figure Lengend Snippet: BH4‐induced pERK1/2 levels in rat dorsal root ganglion neurons are not dependent on nitric oxide synthases. (A–C) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH4 (100 μM) after treatment with the indicated nitric oxide synthase (NOS) inhibitors. Statistical testing by one‐way ANOVA with Bonferroni multiple comparison testing indicated no significant difference between the 0 μM compound and the increasing compound conditions within the PBS (grey) or BH4 (red) treated groups ( p > 0.05). Data presented as individual data points representing biological replicates and mean ± SD (A: F (5, 12) = 0.05394, p = 0.9977; B: F (2, 6) = 0.1033, p = 9034; C: F (2, 9) = 0.04192, p = 0.9591). Experiments have been performed with independent DRG preparations from n = 3 or 4 animals.

Article Snippet: For stimulations, BH4 (Sigma, cat. no. T4425), NGF (Alomone labs, cat. no. N‐240), OSM (Peprotech, cat. no. 400‐36), ascorbic acid (Sigma, cat. no. A4403), phenyl‐alpha‐tert‐butyl nitrone (PBN) (Enzo, cat. no. ALX‐430‐082‐G001), BH2 (MCE MedChemExpress, cat. no. HY‐W008646), H 2 O 2 (Applichem, cat. no. A2726,1000), catalase (Sigma, cat. no. C30‐100MG), superoxide dismutase (Sigma, cat. no. S9697‐15KU), 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol) (Selleckchem, cat. no. S2910), ML171 (also 2‐APT) (Tocris, cat. no. 4653), AITC (Sigma, cat. no. 377430), capsaicin (Sigma, cat. no. M2028), L‐NAME (Cayman, 51298‐62‐5, cat. no. 80210), L‐NNA (Tocris, cat. no. 0664), L‐NMMA (Tocris, cat. no. 0771), ruthenium red (Abcam, cat. no. ab120264), 2‐Aminoethyl diphenylborinate (2‐APB) (Calbiochem, cat. no. 100065), EGTA (Sigma, cat. no. E4378), BAPTA‐AM (Abcam, cat. no. ab120503), Trametinib (ApexBio, cat. no. A3018), U0126 (Calbiochem, cat. no. 662005), Lifirafenib (Chemietek, cat. no. CT‐BGB283), SB590855 (Axonmedchem, cat. no. Axon 2504), GDC0879 (Selleckchem, cat. no. S110), GW5074 (Selleckchem, cat. no. S2872), ZM336372 (Sigma, cat. no. SML0236), Dabrafenib (Selleckchem, cat. no. S2807), Sorafenib (Tocris, cat. no. 6814), Salirasib (Sigma, cat. no. SML1166), Dasatinib (Sigma, cat. no. CDS023389), Saracatinib (Selleckchem, cat. no. S1006), Src inhibitor 1 (Sigma, cat. no. S2075), Edelfosine (cat. no. 3022), U73122 (Tocris, cat. no. 1268), GO6983 (Peprotech, cat. no. 1331975), Thapsigargin (Calbiochem, cat. no. 586005), Ryanodine (Calbiochem, cat. no. 559276), Dantrolene (Tocris, cat. no. 507), JTV‐519 (Sigma, cat. no. SML0549), Heparin (Tocris, cat. no. 2812), Flufenamic acid (Tocris, cat. no. 4522), N‐(p‐amylcinnamoyl)anthranilic acid (ACAA) (Abcam, cat. no. ab141555), M‐8B (Tocris, cat. no. 5324), STO‐609 (Tocris, cat. no. 1551), KN‐93 (Calbiochem, cat. no. 422708), and Autocamtide‐2‐Related Inhibitory Peptide (AIP) (Calbiochem, cat. no. 189485) were used.

Techniques: Ex Vivo, Cell Culture, Comparison

Journal: Journal of Neurochemistry

Article Title: BH4 Oxidation‐Derived H 2 O 2 Activates ERK1 /2 Signaling via B‐Raf in Rat Dorsal Root Ganglion Neurons

doi: 10.1111/jnc.70271

Figure Lengend Snippet: Oxidation‐derived reactive oxygen species like H 2 O 2 and not BH4 itself or BH2 drive ERK1/2 signaling in rat dorsal root ganglion neurons following treatment with BH4. (A) Schematic illustrating the generation of 7,8‐dihydrobiopterin (BH2) and H 2 O 2 by BH4 oxidation. (B) Dose–response curve of relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH4 with or without ascorbic acid (100 μM). Statistical significance tested by comparing fitted curves in a nonlinear regression model (three‐parameter, standard Hill slope). Data presented as mean ± SD. F (3, 76) = 57.83, p < 0.0001 (C, D) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH4 (100 μM) after treatment with the indicated antioxidizing compounds. (E) Dose–response curve of relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH2. Data presented as mean ± SD. (F) Colorimetric analysis of H 2 O 2 levels generated by 100 μM BH4 in PBS using the Enzo hydrogen peroxide chemiluminescent detection kit following manufacturer's instructions with independent H 2 O 2 standard curves (0–100 μM H 2 O 2 ) for each replicate. Data presented as individual data points representing biological replicates and mean ± SD. (G) Dose–response curve of relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to H 2 O 2 . Data presented as mean ± SD, and curve‐fitted line in a nonlinear regression model (three‐parameter, standard Hill slope). (H) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to increasing concentrations of H 2 O 2 or PBS as a control for up to 120 min. Statistical significance tested between H 2 O 2 exposure and PBS control by two‐way ANOVA with Bonferroni's test (* p < 0.05; F (6, 125) = 39.22, p < 0.0001). Data presented as mean ± SD. (I, J) Cell density plots showing single cell data of relative pERK1/2 intensity versus relative RIIβ intensity (whole‐cell average set to 1) (I), or versus soma size (J) from neurons exposed to 25 μM H 2 O 2 . (K, L) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to H 2 O 2 (25 μM) or PBS after treatment with the indicated antioxidizing compounds. (C, D, K, L) Statistical significance tested by one‐way ANOVA with Bonferroni multiple comparison testing, in comparison to 0 μM compound (* p < 0.05). Data presented as individual data points representing biological replicates and mean ± SD. (C: F (2, 9) = 19.03, p = 0.0006; D: F (2, 9) = 11.41, p = 0.0034; K: F (2, 9) = 95.40, p < 0.0001; L: F (2, 9) = 42.13, p < 0.0001). (B–E, G–L) Experiments have been performed with independent DRG preparations from n = 4 animals.

Article Snippet: For stimulations, BH4 (Sigma, cat. no. T4425), NGF (Alomone labs, cat. no. N‐240), OSM (Peprotech, cat. no. 400‐36), ascorbic acid (Sigma, cat. no. A4403), phenyl‐alpha‐tert‐butyl nitrone (PBN) (Enzo, cat. no. ALX‐430‐082‐G001), BH2 (MCE MedChemExpress, cat. no. HY‐W008646), H 2 O 2 (Applichem, cat. no. A2726,1000), catalase (Sigma, cat. no. C30‐100MG), superoxide dismutase (Sigma, cat. no. S9697‐15KU), 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol) (Selleckchem, cat. no. S2910), ML171 (also 2‐APT) (Tocris, cat. no. 4653), AITC (Sigma, cat. no. 377430), capsaicin (Sigma, cat. no. M2028), L‐NAME (Cayman, 51298‐62‐5, cat. no. 80210), L‐NNA (Tocris, cat. no. 0664), L‐NMMA (Tocris, cat. no. 0771), ruthenium red (Abcam, cat. no. ab120264), 2‐Aminoethyl diphenylborinate (2‐APB) (Calbiochem, cat. no. 100065), EGTA (Sigma, cat. no. E4378), BAPTA‐AM (Abcam, cat. no. ab120503), Trametinib (ApexBio, cat. no. A3018), U0126 (Calbiochem, cat. no. 662005), Lifirafenib (Chemietek, cat. no. CT‐BGB283), SB590855 (Axonmedchem, cat. no. Axon 2504), GDC0879 (Selleckchem, cat. no. S110), GW5074 (Selleckchem, cat. no. S2872), ZM336372 (Sigma, cat. no. SML0236), Dabrafenib (Selleckchem, cat. no. S2807), Sorafenib (Tocris, cat. no. 6814), Salirasib (Sigma, cat. no. SML1166), Dasatinib (Sigma, cat. no. CDS023389), Saracatinib (Selleckchem, cat. no. S1006), Src inhibitor 1 (Sigma, cat. no. S2075), Edelfosine (cat. no. 3022), U73122 (Tocris, cat. no. 1268), GO6983 (Peprotech, cat. no. 1331975), Thapsigargin (Calbiochem, cat. no. 586005), Ryanodine (Calbiochem, cat. no. 559276), Dantrolene (Tocris, cat. no. 507), JTV‐519 (Sigma, cat. no. SML0549), Heparin (Tocris, cat. no. 2812), Flufenamic acid (Tocris, cat. no. 4522), N‐(p‐amylcinnamoyl)anthranilic acid (ACAA) (Abcam, cat. no. ab141555), M‐8B (Tocris, cat. no. 5324), STO‐609 (Tocris, cat. no. 1551), KN‐93 (Calbiochem, cat. no. 422708), and Autocamtide‐2‐Related Inhibitory Peptide (AIP) (Calbiochem, cat. no. 189485) were used.

Techniques: Derivative Assay, Ex Vivo, Cell Culture, Generated, Control, Comparison

Journal: Journal of Neurochemistry

Article Title: BH4 Oxidation‐Derived H 2 O 2 Activates ERK1 /2 Signaling via B‐Raf in Rat Dorsal Root Ganglion Neurons

doi: 10.1111/jnc.70271

Figure Lengend Snippet: Oxidation‐derived H 2 O 2 drives BH4‐induced ERK1/2 signaling in rat dorsal root ganglion neurons. (A–D) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH4 (100 μM) or H 2 O 2 (25 μM) after treatment with the indicated antioxidizing or blocking compounds. Statistical significance tested by one‐way ANOVA with Bonferroni multiple comparison testing, in comparison to 0 μM compound (* p < 0.05). Data presented as individual data points representing biological replicates and mean ± SD. (A: BH4, F (2, 9) = 37.25, p < 0.0001; H 2 O 2 , F (2, 9) = 23.39, p = 0.0003). Experiments have been performed with independent DRG preparations from n = 4 animals.

Article Snippet: For stimulations, BH4 (Sigma, cat. no. T4425), NGF (Alomone labs, cat. no. N‐240), OSM (Peprotech, cat. no. 400‐36), ascorbic acid (Sigma, cat. no. A4403), phenyl‐alpha‐tert‐butyl nitrone (PBN) (Enzo, cat. no. ALX‐430‐082‐G001), BH2 (MCE MedChemExpress, cat. no. HY‐W008646), H 2 O 2 (Applichem, cat. no. A2726,1000), catalase (Sigma, cat. no. C30‐100MG), superoxide dismutase (Sigma, cat. no. S9697‐15KU), 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol) (Selleckchem, cat. no. S2910), ML171 (also 2‐APT) (Tocris, cat. no. 4653), AITC (Sigma, cat. no. 377430), capsaicin (Sigma, cat. no. M2028), L‐NAME (Cayman, 51298‐62‐5, cat. no. 80210), L‐NNA (Tocris, cat. no. 0664), L‐NMMA (Tocris, cat. no. 0771), ruthenium red (Abcam, cat. no. ab120264), 2‐Aminoethyl diphenylborinate (2‐APB) (Calbiochem, cat. no. 100065), EGTA (Sigma, cat. no. E4378), BAPTA‐AM (Abcam, cat. no. ab120503), Trametinib (ApexBio, cat. no. A3018), U0126 (Calbiochem, cat. no. 662005), Lifirafenib (Chemietek, cat. no. CT‐BGB283), SB590855 (Axonmedchem, cat. no. Axon 2504), GDC0879 (Selleckchem, cat. no. S110), GW5074 (Selleckchem, cat. no. S2872), ZM336372 (Sigma, cat. no. SML0236), Dabrafenib (Selleckchem, cat. no. S2807), Sorafenib (Tocris, cat. no. 6814), Salirasib (Sigma, cat. no. SML1166), Dasatinib (Sigma, cat. no. CDS023389), Saracatinib (Selleckchem, cat. no. S1006), Src inhibitor 1 (Sigma, cat. no. S2075), Edelfosine (cat. no. 3022), U73122 (Tocris, cat. no. 1268), GO6983 (Peprotech, cat. no. 1331975), Thapsigargin (Calbiochem, cat. no. 586005), Ryanodine (Calbiochem, cat. no. 559276), Dantrolene (Tocris, cat. no. 507), JTV‐519 (Sigma, cat. no. SML0549), Heparin (Tocris, cat. no. 2812), Flufenamic acid (Tocris, cat. no. 4522), N‐(p‐amylcinnamoyl)anthranilic acid (ACAA) (Abcam, cat. no. ab141555), M‐8B (Tocris, cat. no. 5324), STO‐609 (Tocris, cat. no. 1551), KN‐93 (Calbiochem, cat. no. 422708), and Autocamtide‐2‐Related Inhibitory Peptide (AIP) (Calbiochem, cat. no. 189485) were used.

Techniques: Derivative Assay, Ex Vivo, Cell Culture, Blocking Assay, Comparison

Journal: Journal of Neurochemistry

Article Title: BH4 Oxidation‐Derived H 2 O 2 Activates ERK1 /2 Signaling via B‐Raf in Rat Dorsal Root Ganglion Neurons

doi: 10.1111/jnc.70271

Figure Lengend Snippet: Antioxidizing capacities of ruthenium red, 2‐APB, and BAPTA‐AM interfere with the analysis of a potential Ca 2+ ‐dependency of BH4‐ or H 2 O 2 ‐induced ERK1/2 signaling in rat dorsal root ganglion neurons. (A–D) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH4 (100 μM) or H 2 O 2 (25 μM) after treatment with the indicated compounds. Statistical significance tested by one‐way ANOVA with Bonferroni multiple comparison testing (A, B, D) or by unpaired, two‐sided t‐ test (C), in comparison to 0 μM compound (* p < 0.05). Data presented as individual data points representing biological replicates and mean ± SD. Experiments have been performed with independent DRG preparations from n = 4–5 animals depicted by individual data points. (E) Colorimetric analysis of total antioxidant capacity of the indicated reagents in PBS using the Total Antioxidant Capacity Colorimetric Assay Kit, following manufacturer's instructions with independent Trolox standard curves (0–1.43 mM) for each replicate. Data presented as individual data points representing biological replicates and mean ± SD. (A–E) Data presented as individual data points representing biological replicates and mean ± SD (A: BH4, F (2, 9) = 14.08, p = 0.0007; H 2 O 2 , F (2, 9) = 6.612, p = 0.0116; B: BH4, F (2, 9) = 18.37, p = 0.0007; H 2 O 2 , F (2, 9) = 50.90, p < 0.0001; D: BH4, F (2, 9) = 12.07, p = 0.0013; H 2 O 2 , F (2, 12) = 5.161, p = 0.0241).

Article Snippet: For stimulations, BH4 (Sigma, cat. no. T4425), NGF (Alomone labs, cat. no. N‐240), OSM (Peprotech, cat. no. 400‐36), ascorbic acid (Sigma, cat. no. A4403), phenyl‐alpha‐tert‐butyl nitrone (PBN) (Enzo, cat. no. ALX‐430‐082‐G001), BH2 (MCE MedChemExpress, cat. no. HY‐W008646), H 2 O 2 (Applichem, cat. no. A2726,1000), catalase (Sigma, cat. no. C30‐100MG), superoxide dismutase (Sigma, cat. no. S9697‐15KU), 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol) (Selleckchem, cat. no. S2910), ML171 (also 2‐APT) (Tocris, cat. no. 4653), AITC (Sigma, cat. no. 377430), capsaicin (Sigma, cat. no. M2028), L‐NAME (Cayman, 51298‐62‐5, cat. no. 80210), L‐NNA (Tocris, cat. no. 0664), L‐NMMA (Tocris, cat. no. 0771), ruthenium red (Abcam, cat. no. ab120264), 2‐Aminoethyl diphenylborinate (2‐APB) (Calbiochem, cat. no. 100065), EGTA (Sigma, cat. no. E4378), BAPTA‐AM (Abcam, cat. no. ab120503), Trametinib (ApexBio, cat. no. A3018), U0126 (Calbiochem, cat. no. 662005), Lifirafenib (Chemietek, cat. no. CT‐BGB283), SB590855 (Axonmedchem, cat. no. Axon 2504), GDC0879 (Selleckchem, cat. no. S110), GW5074 (Selleckchem, cat. no. S2872), ZM336372 (Sigma, cat. no. SML0236), Dabrafenib (Selleckchem, cat. no. S2807), Sorafenib (Tocris, cat. no. 6814), Salirasib (Sigma, cat. no. SML1166), Dasatinib (Sigma, cat. no. CDS023389), Saracatinib (Selleckchem, cat. no. S1006), Src inhibitor 1 (Sigma, cat. no. S2075), Edelfosine (cat. no. 3022), U73122 (Tocris, cat. no. 1268), GO6983 (Peprotech, cat. no. 1331975), Thapsigargin (Calbiochem, cat. no. 586005), Ryanodine (Calbiochem, cat. no. 559276), Dantrolene (Tocris, cat. no. 507), JTV‐519 (Sigma, cat. no. SML0549), Heparin (Tocris, cat. no. 2812), Flufenamic acid (Tocris, cat. no. 4522), N‐(p‐amylcinnamoyl)anthranilic acid (ACAA) (Abcam, cat. no. ab141555), M‐8B (Tocris, cat. no. 5324), STO‐609 (Tocris, cat. no. 1551), KN‐93 (Calbiochem, cat. no. 422708), and Autocamtide‐2‐Related Inhibitory Peptide (AIP) (Calbiochem, cat. no. 189485) were used.

Techniques: Ex Vivo, Cell Culture, Comparison, Colorimetric Assay

Journal: Journal of Neurochemistry

Article Title: BH4 Oxidation‐Derived H 2 O 2 Activates ERK1 /2 Signaling via B‐Raf in Rat Dorsal Root Ganglion Neurons

doi: 10.1111/jnc.70271

Figure Lengend Snippet: BH4 and H 2 O 2 induce increased pERK1/2 levels in rat dorsal root ganglion neurons via B‐Raf and MEK. (A) Schematic illustrating relevant components of the ERK1/2 signaling cascade in DRG neurons that were analyzed in this study (PKC, protein kinase C; PLC, phospholipase C; RTKs, receptor tyrosine kinases; SRC, Src kinases). (B) Table listing all compounds used to interfere with ERK1/2 signaling. (C–K) Relative pERK1/2 intensity in ex vivo cultured rat DRG neurons (UCHL1 + ) exposed to BH4 (100 μM) or H 2 O 2 (25 μM) after treatment with the indicated compounds. Statistical significance tested by one‐way ANOVA with Bonferroni multiple comparison testing, in comparison to 0 μM compound (* p < 0.05). Data presented as individual data points representing biological replicates and mean ± SD. (C: BH4, F (2, 9) = 14.08, p = 0.0007; H 2 O 2 , F (2, 9) = 6.612, p = 0.0116; D: BH4, F (2, 9) = 9.276, p = 0.0065; H 2 O 2 , F (2, 6) = 10.60, p = 0.0107; F: BH4, F (2, 9) = 19.42, p = 0.0005; H 2 O 2 , F (2, 12) = 75.96, p < 0.0001; G: BH4, F (2, 9) = 11.19, p = 0.0036; H 2 O 2 , F (2, 9) = 126.8, p < 0.0001; J: BH4, F (2, 9) = 26.96, p = 0.0002; H 2 O 2 , F (2, 6) = 65.74, p < 0.0001). Experiments have been performed with independent DRG preparations from n = 3–4 animals, depicted by individual data points.

Article Snippet: For stimulations, BH4 (Sigma, cat. no. T4425), NGF (Alomone labs, cat. no. N‐240), OSM (Peprotech, cat. no. 400‐36), ascorbic acid (Sigma, cat. no. A4403), phenyl‐alpha‐tert‐butyl nitrone (PBN) (Enzo, cat. no. ALX‐430‐082‐G001), BH2 (MCE MedChemExpress, cat. no. HY‐W008646), H 2 O 2 (Applichem, cat. no. A2726,1000), catalase (Sigma, cat. no. C30‐100MG), superoxide dismutase (Sigma, cat. no. S9697‐15KU), 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol) (Selleckchem, cat. no. S2910), ML171 (also 2‐APT) (Tocris, cat. no. 4653), AITC (Sigma, cat. no. 377430), capsaicin (Sigma, cat. no. M2028), L‐NAME (Cayman, 51298‐62‐5, cat. no. 80210), L‐NNA (Tocris, cat. no. 0664), L‐NMMA (Tocris, cat. no. 0771), ruthenium red (Abcam, cat. no. ab120264), 2‐Aminoethyl diphenylborinate (2‐APB) (Calbiochem, cat. no. 100065), EGTA (Sigma, cat. no. E4378), BAPTA‐AM (Abcam, cat. no. ab120503), Trametinib (ApexBio, cat. no. A3018), U0126 (Calbiochem, cat. no. 662005), Lifirafenib (Chemietek, cat. no. CT‐BGB283), SB590855 (Axonmedchem, cat. no. Axon 2504), GDC0879 (Selleckchem, cat. no. S110), GW5074 (Selleckchem, cat. no. S2872), ZM336372 (Sigma, cat. no. SML0236), Dabrafenib (Selleckchem, cat. no. S2807), Sorafenib (Tocris, cat. no. 6814), Salirasib (Sigma, cat. no. SML1166), Dasatinib (Sigma, cat. no. CDS023389), Saracatinib (Selleckchem, cat. no. S1006), Src inhibitor 1 (Sigma, cat. no. S2075), Edelfosine (cat. no. 3022), U73122 (Tocris, cat. no. 1268), GO6983 (Peprotech, cat. no. 1331975), Thapsigargin (Calbiochem, cat. no. 586005), Ryanodine (Calbiochem, cat. no. 559276), Dantrolene (Tocris, cat. no. 507), JTV‐519 (Sigma, cat. no. SML0549), Heparin (Tocris, cat. no. 2812), Flufenamic acid (Tocris, cat. no. 4522), N‐(p‐amylcinnamoyl)anthranilic acid (ACAA) (Abcam, cat. no. ab141555), M‐8B (Tocris, cat. no. 5324), STO‐609 (Tocris, cat. no. 1551), KN‐93 (Calbiochem, cat. no. 422708), and Autocamtide‐2‐Related Inhibitory Peptide (AIP) (Calbiochem, cat. no. 189485) were used.

Techniques: Ex Vivo, Cell Culture, Comparison