Journal: Neural Regeneration Research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Effects of thrombin/protease-activated receptor 1 on the mitogen-activated protein kinase (MAPK)/nuclear factor kappa-B (NFκB) pathway in astrocytes. (A) Western blot analysis of phosphorylated extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), P38 kinase (P38), and NFκB protein levels following astrocyte stimulation with 0, 0.5, 1, or 2 U/mL thrombin for 24 hours. (B–E) Quantification of phosphorylated ERK, JNK, P38, and NFκB protein levels, normalized to β-actin. (F) Western blot analysis of phosphorylated ERK, JNK, P38, and NFκB protein levels following astrocyte stimulation with 1 U/mL thrombin in the presence of 0–3 μM SCH79797 for 24 hours. (G–J) Quantification of phosphorylated ERK, JNK, P38, and NFκB protein levels, normalized to β-actin. Data are expressed as mean ± SEM ( n = 3). * P < 0.05 (one-way analysis of variance followed by Bonferroni’s post hoc comparison test). SCH79797: Protease-activated receptor 1 inhibitor.

Article Snippet: After blocking with 5% skim milk for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: CH25H (rabbit, 1:500, Invitrogen, Cat# PA5-70691, RRID: AB_2689560), phosphorylated extracellular signal regulated kinase (pERK)1/2 (rabbit, 1:1000, CST, Cat# 9102S, RRID: AB_330744), extracellular signal regulated kinase (ERK)1/2 (rabbit, 1:1000, CST, Cat# 8544S, RRID: AB_11127856), phosphorylated c-Jun N-terminal kinase (pJNK; rabbit, 1:1000, CST, Cat# 9251S, RRID: AB_331659), JNK (rabbit, 1:1000, CST, Cat# 9252S, RRID: AB_2250373), phosphorylated P38 kinase (pP38; rabbit, 1:1000, CST, Cat# 8632S, RRID: AB_2797648), P38 (rabbit, 1:1000, CST, Cat# 14451S, RRID: AB_2798482), nuclear factor kappa-B (NFκB; rabbit, 1:1000, CST, Cat# 12629S, RRID: AB_2722509), and β-actin (mouse, 1:5000, CST, Cat# 3700S, RRID: AB_2242334).

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Effects of mitogen-activated protein kinase (MAPK) or nuclear factor kappa-B (NFκB) inhibitors on astrocyte cholesterol-25-hydroxylase (CH25H) expression in response to stimulation with thrombin. (A) Western blot analysis of NFκB expression following treatment with 1 U/mL thrombin in the presence of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 hours. (B) Quantification of NFκB protein expression, normalized to β-actin. (C) Western blot analysis of CH25H expression following treatment with 1 U/mL thrombin in the presence of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 hours. (D) Quantification of CH25H expression, normalized to β-actin. (E) Western blot analysis of CH25H expression following treatment with 1 U/mL thrombin in the presence or absence of 10–100 μM of the NFκB inhibitor PDTC for 24 hours. (F) Quantification of CH25H expression, normalized to β-actin. Data are expressed as mean ± SEM ( n = 3). * P < 0.05 (one-way analysis of variance followed by Bonferroni’s post hoc comparison test). DMSO: Dimethyl sulfoxide; PD98059: extracellular regulated protein kinases inhibitor; SB203580: P38 inhibitor; SP600125: c-Jun N-terminal kinase inhibitor.

Article Snippet: After blocking with 5% skim milk for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: CH25H (rabbit, 1:500, Invitrogen, Cat# PA5-70691, RRID: AB_2689560), phosphorylated extracellular signal regulated kinase (pERK)1/2 (rabbit, 1:1000, CST, Cat# 9102S, RRID: AB_330744), extracellular signal regulated kinase (ERK)1/2 (rabbit, 1:1000, CST, Cat# 8544S, RRID: AB_11127856), phosphorylated c-Jun N-terminal kinase (pJNK; rabbit, 1:1000, CST, Cat# 9251S, RRID: AB_331659), JNK (rabbit, 1:1000, CST, Cat# 9252S, RRID: AB_2250373), phosphorylated P38 kinase (pP38; rabbit, 1:1000, CST, Cat# 8632S, RRID: AB_2797648), P38 (rabbit, 1:1000, CST, Cat# 14451S, RRID: AB_2798482), nuclear factor kappa-B (NFκB; rabbit, 1:1000, CST, Cat# 12629S, RRID: AB_2722509), and β-actin (mouse, 1:5000, CST, Cat# 3700S, RRID: AB_2242334).

Techniques: Expressing, Western Blot

Journal: Nature Communications

Article Title: Cisplatin toxicity is counteracted by the activation of the p38/ATF-7 signaling pathway in post-mitotic C. elegans

doi: 10.1038/s41467-023-38568-5

Figure Lengend Snippet: a Western blot analysis after SDS-PAGE of phospho-p38 levels in 1-day-old wild-type adults with and without cisplatin treatment for indicated times. Worms were exposed to cisplatin on 300 μg/mL cisplatin-containing plates. The blot was probed with anti:phospho-p38 antibody to detect activated PMK-1, anti:PMK-1 antibody was used to estimate total levels of PMK-1 protein, tubulin was used as a loading control. Full uncropped images are available in Supplementary Fig. . b Relative phospho-p38 level quantification from blots presented in Supplementary Fig. . Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. Bars represent mean ± SD. c Western blot analysis after SDS-PAGE of phospho-p38 levels in 1-day-old wild-type adults with (+) and without (−) MitoTempo/cisplatin exposure. The blot was probed with anti:phospho-p38 antibody, tubulin was used as a loading control. Full uncropped images are available in Supplementary Fig. . d Relative phospho-p38 levels quantification from blots presented in Supplementary Fig. . Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. Bars represent mean ± SD. e , h , i Mean survival ± SD of 1-day-old adults with the indicated genotypes after 24 h cisplatin (300 μg/mL) exposure. Statistical significance determined was by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. All data on cisplatin sensitivity trials is available in Supplementary Data . f , g Relative gene expression of four phase II detoxification genes in 1-day-old adult wild-type animals ( f ) without or with (+CP) 6 h or 24 h cisplatin treatment, g without or with (+CP) 6 h cisplatin treatment. Worms were exposed to cisplatin on 300 μg/mL cisplatin-containing plates. Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate for 6 h exposure timepoint and duplicate for 24 h exposure timepoint. F44B9.5 was used as a normalizing control. Bars represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were: anti:phospho-p38 MAPK (Thr180/Thr182) Rabbit mAb (D3F9, Cell Signaling) 1:1000, anti:total PMK-1 MAPK Rabbit pAb 1:1000 from the Pukkila-Worley lab , anti:tubulin (T5168, Sigma) 1:5000, anti:Flag (M2, Sigma) 1:1000.

Techniques: Western Blot, SDS Page, Expressing

Journal: Nature Communications

Article Title: Cisplatin toxicity is counteracted by the activation of the p38/ATF-7 signaling pathway in post-mitotic C. elegans

doi: 10.1038/s41467-023-38568-5

Figure Lengend Snippet: a Western blot analysis after SDS-PAGE of phospho-p38 levels in 1-day-old adults with (+) and without (–) 1 mM auxin (AUX) treatment. The blot was probed with anti:phospho-p38 antibody and tubulin was used as a loading control. Full uncropped images are available in Supplementary Fig. . b , d Mean survival ± SD of 1-day-old adults with the indicated genotypes after 24 h exposure to 300 μg/mL cisplatin-containing plates without (−AUX) or with (+AUX) 1 mM auxin treatment (see the “Methods” section for details). Statistical significance was determined by the b independent two-sided t -test or d one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. c Western blot analysis after SDS-PAGE of SEK-1 levels in 1-day-old adults with (+) and without (−) 1 mM auxin (AUX) treatment. The blot was probed with anti:Flag antibody and tubulin was used as a loading control. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were: anti:phospho-p38 MAPK (Thr180/Thr182) Rabbit mAb (D3F9, Cell Signaling) 1:1000, anti:total PMK-1 MAPK Rabbit pAb 1:1000 from the Pukkila-Worley lab , anti:tubulin (T5168, Sigma) 1:5000, anti:Flag (M2, Sigma) 1:1000.

Techniques: Western Blot, SDS Page

Journal: Nature Communications

Article Title: ERK and USP5 govern PD-1 homeostasis via deubiquitination to modulate tumor immunotherapy

doi: 10.1038/s41467-023-38605-3

Figure Lengend Snippet: a – c IB analysis of WCL derived from HEK293T cells co-transfected PD-1-cFlag or PD-1-cHA with different kinases as indicated. d Representative mIHC images of CD3 (pink), PD-1 (red), phosphor-ERK at Thr202/Try204 (p-ERK, green), CK (cyan), and DAPI nuclear staining (blue) in human colon tumor sections. White arrows indicate positive cells for PD-1 and p-ERK colocalization. Yellow color is considered overlapping for PD-1 and p-ERK staining. n = 5. Scale bars, 50 μm. e IB analysis of WCL derived from Jurkat cells treated with PHA (150 ng/mL) for 3 days and Trametinib (1 or 3 μM) for 24 h before harvesting. f , g Cell surface PD-1 on shGFP- or shERK1/2-treated Jurkat cells with pre-stimulation of PHA (150 ng/mL) for 3 days was analyzed by flow cytometry ( f ). The relative mean fluorescence intensity (MFI) of PD-1 on the surface of was quantified ( g ). Data were presented as mean ± S.D. n = 3 biologically independent samples per group. Two-tailed unpaired t -test. h A schematic illustration and sequence alignment of a potential ERK binding D-domain on PD-1 protein sequence, (K/R) 0-2 -(X) 1-6 -Φ-X-Φ, where Φ is a hydrophobic residue and X is any amino acid. i, j IB analysis of WCL and anti-HA IPs ( i ) or GST pull-down precipitates ( i ) from HEK293T cell lysates transfected with indicated constructs. k , l In vitro phosphorylation assays of bacterially purified recombinant GST, GST-PD-1 truncations or T234A mutant by ERK1 kinase. m , n IB analysis of WCL and anti-Flag or anti-HA IPs derived from HEK293T cells transfected with indicated constructs. o – q IB analysis of WCL and anti-PD-1 IPs from Jurkat pre-treated with PHA (150 ng/mL) for 3 days ( o , p ) or MOLT-4 ( q ) cells. Cells were treated with indicated Trametinib (0.5 or 1 μM) for 24 h ( p , q ). All IB data are representative of two independent experiments. Source data are provided as a Source data file.

Article Snippet: Anti-PD-1 (D4W2J) rabbit mAb (1:2000 dilution, Cat# 86163, RRID:AB_2728833), anti-PD-1 (Intracellular Domain) (D7D5W) rabbit mAb (1:2000 dilution, Cat# 84651, RRID:AB_2800041), anti-K48-linkage Specific Polyubiquitin rabbit pAb (1:1000 dilution, Cat# 4289, RRID:AB_10557239), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (1:2000 dilution, Cat# 4370, RRID:AB_2315112), anti-Myc-Tag (71D10) Rabbit mAb (1:1000 dilution, Cat# 2278, RRID:AB_490778), and anti-USP9X (D4Y7W) rabbit mAb (1:2000 dilution, Cat# 14898, RRID:AB_2798640) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Transfection, Staining, Flow Cytometry, Fluorescence, Two Tailed Test, Sequencing, Binding Assay, Construct, In Vitro, Purification, Recombinant, Mutagenesis

Journal: Nature Communications

Article Title: ERK and USP5 govern PD-1 homeostasis via deubiquitination to modulate tumor immunotherapy

doi: 10.1038/s41467-023-38605-3

Figure Lengend Snippet: a – c IB analysis of WCL and IPs from HEK293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 12 h. d , e IB analysis of WCL and anti-HA IPs from HEK293T cells transfected with indicated constructs ( d ) or anti-PD-1 IPs derived from MOTL-4 cells ( e ). Cells were treated with indicated Trametinib (0.5 or 1 μM) for 24 h and 10 μM MG132 for 12 h ( d ) or 4 h ( e ). f IB analysis of WCL and GST pull-down precipitates from HEK293T cell lysates with ectopic expression of PD-1-cHA incubated with recombinant GST-USP5 protein. Cells were treated with 1 μM Ulixertinib for 24 h. g IB analysis of WCL and anti-HA IPs from HEK293T cells transfected with indicted USP5, PD-1 WT, or T234A mutant. Cells were treated with 10 μM MG132 for 12 h. h IB analysis of WCL and GST pull-down precipitates from HEK293T cell lysates with ectopic expression of PD-1-cHA WT or PD-1-cHA T234A incubated with bacterially purified recombinant GST-USP5 protein. i Bacterially purified recombinant GST-PD-1 (192-288) WT or T234A mutant was phosphorylated by ERK1 in vitro. Subsequently, GST pull-down assay was performed with purified recombinant His-USP5. IB analysis with indicated antibodies. j , k 3 μg of indicated biotin-labeled synthetic PD-1 peptides were incubated with 4 μg purified recombinant GST-USP5 ( j ) or different domains ( k ), respectively. Streptavidin beads were added to perform pull-down assays and precipitations were analyzed with IB as indicated. Dot blot was used to identify biotin-labeled synthetic PD-1 peptides. l , m IB analysis of WCL and Ni-NTA pull-down products from lysates of HEK293T cells transfected with the indicated constructs and treated with/without Trametinib (1 or 3 μM) for 12 h ( l ). Cells were treated with 10 μM MG132 for 12 h. n IB analysis of WCL and anti-PD-1 denatured-IPs from lysates of shGFP- or shERK1/2-treated Jurkat cells using indicated antibodies. Cells were pre-treated with PHA (150 ng/mL) for 3 days and 5 μM MG132 for 6 h. All data are representative of two independent experiments. Source data are provided as a Source data file.

Article Snippet: Anti-PD-1 (D4W2J) rabbit mAb (1:2000 dilution, Cat# 86163, RRID:AB_2728833), anti-PD-1 (Intracellular Domain) (D7D5W) rabbit mAb (1:2000 dilution, Cat# 84651, RRID:AB_2800041), anti-K48-linkage Specific Polyubiquitin rabbit pAb (1:1000 dilution, Cat# 4289, RRID:AB_10557239), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (1:2000 dilution, Cat# 4370, RRID:AB_2315112), anti-Myc-Tag (71D10) Rabbit mAb (1:1000 dilution, Cat# 2278, RRID:AB_490778), and anti-USP9X (D4Y7W) rabbit mAb (1:2000 dilution, Cat# 14898, RRID:AB_2798640) were purchased from Cell Signaling Technology.

Techniques: Transfection, Construct, Derivative Assay, Expressing, Incubation, Recombinant, Mutagenesis, Purification, In Vitro, Pull Down Assay, Labeling, Dot Blot

Journal: Journal of Inflammation (London, England)

Article Title: Dioscin alleviates the progression of osteoarthritis: an in vitro and in vivo study

doi: 10.1186/s12950-023-00339-w

Figure Lengend Snippet: Dio blocks IL-1β-induced MAPK signaling pathway activation. Chondrocytes were exposed to IL-1β (10 ng/mL) with or without Dio (200, 400, and 800 ng/mL) for 30 min. A Representative western blots and ( B ) quantitative analysis of P38, P-P38, ERK, P-ERK, JNK, and P-JNK in each group. # P < 0.05 vs. control group; * P < 0.05 vs. IL-1β group; ** P < 0.01 vs. IL-1β group; n = 3

Article Snippet: Antibodies specific for P65 (#8242), phosphorylated P65 (P-P65) (#3033), IκBα (#4814), phosphorylated-IκBα (P-IκBα) (#2859), IKKβ (#8943), phosphorylated-IKKα/β (P-IKKα/β) (#2697), P38 (#8690), phosphorylated-p38 (p-p38) (#4511), ERK (#4695), phosphorylated-ERK (P-ERK) (#4370), JNK (#9252), and phosphorylated-JNK (P-JNK) (#4668) were procured from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Western Blot