pmkk4  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Remyelination protects neurons from DLK-mediated neurodegeneration

doi: 10.1038/s41467-024-53429-5

Figure Lengend Snippet: a Experimental schematic and images of laser microdissection of the GCL of the retina. b Heatmap of expression of RGC-specific transcripts in the micro-dissected GCL samples relative to whole retina. c Heatmap of select transcripts from the GCL known to be activated by c-Jun/DLK signaling compared between genotypes. In situ hybridization in the retina at 10 weeks post tamoxifen with probes against Rbpms and Hrk ( d ) or Ecel1 ( e ). Arrowheads indicate double-positive cells. f Retina at 10 weeks post tamoxifen stained with phosphorylated (Ser63) c-Jun. g Quantification of phosphorylated c-Jun cells in the retinae of each genotype. **** p < 0.0001, and Myrf ΔiPlp1 relative to Myrf fl/fl at 10 (** p = 0.0088) and 20 weeks post tamoxifen (** p = 0.0092). Week 10 Myrf fl/fl n = 16, Myrf ΔiPlp1 n = 4, Myrf ΔiSox10 n = 8, week 12 Myrf fl/fl n = 7, Myrf ΔiPlp1 n = 3, Myrf ΔiSox10 n = 3, and week 20 Myrf fl/fl n = 5, Myrf ΔiPlp1 n = 7 mice. h Phosphorylated c-Jun costained with RBPMS cells in Myrf ΔiSox10 mice. Inlays are of boxed area. Arrowheads indicate colabeled cells. i Representative images of phosphorylated c-Jun stained with AP-2α/β in Myrf ΔiSox10 retinae. Inlays are of boxed area. Arrows indicate phosphorylated c-Jun positive cells negative for AP-2α/β. j Quantification of RBPMS and AP-2α/β expression in phosphorylated c-Jun+ cells. k Image of retinal layers stained with phosphorylated c-Jun, RBPMS and DAPI. Inlays are of boxed area. l Schematic of the DLK-mediated MAPK cascade and c-Jun. m Western blot of optic nerves for DLK, pMKK4, MKK4, pJNK, JNK, MOG and β-actin loading control from optic nerves of Myrf fl/fl , Myrf ΔiPlp1 and Myrf ΔiSox10 mice. n Quantification of western blots in Myrf ΔiPlp1 mice relative to Myrf fl/fl . *** p = 0.0007 and * p = 0.0309. n = 4 per group. o Quantification of western blots in Myrf ΔiSox10 mice relative to Myrf fl/fl . pMKK4 (* p = 0.0434), pJNK (** p = 0.0072), total JNK (** p = 0.0034) and MOG ** p = 0.0038). Myrf fl/fl n = 4 and Myrf ΔiSox10 n = 3. Scale bars are 50 µm in ( a , f , h , i , k ), and 5 µm in ( d , e ), insets in ( h , i , k ). Two-way ANOVA with Tukey’s post hoc at 10 and 12 weeks post tamoxifen, and Student’s t test to compare groups at 20 weeks post tamoxifen in ( g ). Student’s t test in ( n , o ). All statistical tests are two-sided. Error bars are SEM. NA not applicable. Source data for this Figure are provided as a Source Data file. Schematic in a created in BioRender. Duncan (2023). BioRender.com/n39a548. l created in BioRender. Duncan (2023) BioRender.com/g94u157.

Article Snippet: Following transfer, blots were rinsed in 1x TBS with 0.1% Tween-20 (TBST) before blocking in 1x TBST with 5% milk powder for 1 h. Blots were probed with antibodies against DLK (GTX124127, Genetex), pMKK4 (9156, Cell Signaling), MKK4 (9152, Cell Signaling), pJNK (9251, Cell Signaling), JNK (9252, Cell Signaling), MOG (supernatant from clone 8-18C5, kind gift of R. Reynolds, Imperial College, London, UK), MBP (MAB386, Millipore) diluted in TBST with 2% BSA (BP9706-100, Fisher Scientific) overnight to 1:1000.

Techniques: Laser Capture Microdissection, Expressing, In Situ Hybridization, Staining, Western Blot, Control

Journal: Food & function

Article Title: Hexane insoluble fraction from purple rice extract improves steatohepatitis and fibrosis via inhibition of NF-κB and JNK signaling.

doi: 10.1039/d4fo00292j

Figure Lengend Snippet: Fig. 6 Hexane insoluble fraction of purple rice ethanolic extract reduces phosphorylated NF-κB and JNK protein levels in rat nonalco- holic steatohepatitis connexin 32 dominant negative transgenic (Cx32ΔTg) rats were fed a contol diet, high-fat diet (HFD), or HFD plus hexane insoluble fraction of purple rice ethanolic extract (PRE-HIF) for 17 weeks. (a) Nuclear factor (NF)-κB-related (NF-κB, PNF-κB, IκB-α) and SAPK/JNK (CDC42, MKK4, PMKK4, JNK, and PJNK) signaling proteins in control, HFD, and HFD + PRE groups by western blotting. Protein samples from individual rats were loaded into each lane. (b) Data are shown as the mean ± sd. #p < 0.05, ###p < 0.001 statistically significant compared with the HFD group.

Article Snippet: Proteins from frozen tissues collected from the left lateral lobe of the liver were extracted as previously outlined.12 The antibodies used were against the following antigens: Cdc42 (Cell Signaling Technology, Danvers, MA, Cat# 2466, RRID: AB_2078082), NF-κB (Cell Signaling Technology Cat# 8242, RRID:AB_10859369), phosphorylated (p) NF-κB (Ser536) (Cell Signaling Technology Cat# 3031, RRID:AB_330559), MKK4 (Cell Signaling Technology Cat# 9152, RRID:AB_330905), pMKK4 (Thr261) (Cell Signaling Technology Cat# 9151, RRID: AB_330889), JNK (Cell Signaling Technology Cat# 9252, RRID: AB_2250373), pJNK (Thr183/Tyr185) (Cell Signaling Technology Cat# 9251, RRID:AB_331659); and β-actin (SigmaAldrich, St Louis, MI, USA, Cat# A5316, RRID:AB_476743).

Techniques: Dominant Negative Mutation, Transgenic Assay, Control, Western Blot

Journal: Nutrients

Article Title: Lactoferrin Prevents Hepatic Injury and Fibrosis via the Inhibition of NF-κB Signaling in a Rat Non-Alcoholic Steatohepatitis Model

doi: 10.3390/nu14010042

Figure Lengend Snippet: Down-regulation of inflammatory cytokines and deactivation of NF-κB and JNK signaling after the administration of lactoferrin in nonalcoholic steatohepatitis induced in Cx32 dominant negative transgenic rats. Connexin 32 dominant negative transgenic (Cx32ΔTg) rats were fed a high-fat diet (HFD), given an intraperitoneal injection of dimethylnitrosamine (DMN), and treated with lactoferrin (LF) for 17 weeks. ( a ) Protein levels of nuclear factor (NF)-κB-related (NF-κB, phosphorylated (p)NF-κB, IκB-α) and SAPK/JNK (Cdc42, Mkk4, pMkk4, Jnk, and pJnk) signaling proteins in Control, LF 100 mg/kg/day (LF100), or LF 500 mg/kg/day (LF500) rat groups were assessed by western blotting. Each lane represents a protein sample from an individual rat. Phospho, phosphorylated. ( b ) Data is shown as the mean ± SD. * p < 0.05, ** p < 0.01 compared to the Control group.

Article Snippet: Membranes were probed with primary antibodies against: Cdc42, IκB-α, NF-κB, phosphorylated (p) NF-κB (Ser536), Mkk4, pMkk4 (Thr261), Jnk, pJnk (Thr183/Tyr185) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MI, USA).

Techniques: Dominant Negative Mutation, Transgenic Assay, Injection, Control, Western Blot