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

Journal: Cancer Cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet:

Article Snippet: Anti-human/mouse pMKK4 [S257] clone C36C11 , Cell Signaling Technology , 4514.

Techniques: Blocking Assay, Virus, Recombinant, Plasmid Preparation, Saline, Lysis, Staining, Reverse Transcription, In Vivo, Electroporation, Mass Cytometry, Conjugation Assay, Illumina Sequencing, Library Quantification, In Vitro, Quantitative RT-PCR, Software, Real-time Polymerase Chain Reaction, Cytometry, Microscopy, Imaging, Spectrophotometry

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A-C, E-G, I) Kdo-2 Lipid A and P3C co-treatment (50 nM each) in iBMDMs. (D, H) Co-TLR (PAMP: Kdo-2 Lipid A and P3C, 50 nM each) or multi-PRR priming (heat-killed Yersinia pseudotuberculosis , MOI 100) followed by Nigericin (10 μ M) trigger for 2h. (A) Quantification of the Pearson’s correlation coefficient (PCC) for the correlation of pJNK antibody staining in IRAK1 clusters. (B) Quantification of the Mander’s colocalization coefficient (MCC) for the co-occurrence of pJNK antibody staining in IRAK1 clusters. (C) pJNK staining in Irak1 −/− iBMDMs. (D) IRAK1-pJNK PLA. (E) PCC and (F) MCC for pMKK7 antibody staining in IRAK1 clusters. (G) pMKK7 staining in Irak1 −/− iBMDMs. (H) IRAK1-pMKK7 PLA in BMDMs. (I) pMKK4 staining in iBMDMs mIRAK1mCherry. (Panels A, B, E, F) Each data point represents value from a randomly selected non-over lapping field of cells. Paired t test. Data are represented as mean ± SD. Automated imaging of at least 2 (Panels C, G, I) fields of cells. At least 20000 (Panels D, H) cells imaged. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Primary – IRAK1 Rb (CST 4504S), IRAK1 Rb (Proteintech 10478-2-AP), IRAK1 Ms (SCBT sc-5288), ASC Rb (SCBT sc-22514-R), ASC Rb (CST 67824S), ASC Ms (SCBT sc-514414), ASC Ms (EMD Millipore 04-147), pASC Rb (ECM Biosciences AP5631), NLRP3 Ms (AdipoGen AG-20B-0014-C100), NLRC4 Rb (EMD Millipore 06-1125), pJNK1/2 Rb (Invitrogen 700031), IRAK4 Rb (CST 4363S), IRAK3 Rb (Thermo Scientific PA5-19969), IRAK2 Rb (Abcam ab66017), IRAK2 Ms (SCBT sc-515885), MyD88 Rb (CST 4283S), MyD88 Gt (R&D Systems AF3109), Rb IgG Isotype (CST 3900S), Ms IgG Isotype (CST 5415S), TRAF6 Rb (Bioss bs-1184R), TRAF6 Rb (Abcam ab33915), pTBK1 Rb (CST 5483S), pJNK Rb (Invitrogen 700031), pMKK4 Rb (CST 4514P), pMKK7 Rb (CST 4171S), p44/42 Rb (CST 4695P), p65 Rb (Abcam ab16502), pATF2 Ms (SCBT sc-8398), pATF2 Rb (CST 5112S), Pellino-1 (CST 31474S), TICAM2 Ms (SCBT sc-376076), pERK5 Rb (CST 3371S), pTAK1 Rb (CST 4531S), pTAB2 Rb (CST 8155S), βTrCP Rb (CST 11984S), Proteasome 20S alpha 5 Rb (Novus Biologicals NBP1-86838).

Techniques: Staining, Imaging