Journal: bioRxiv

Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

doi: 10.1101/738682

Figure Lengend Snippet: Neutrophils were incubated with DMSO, Erbstatin [Erb, 40 µM] (A) or tyrphostin AG825 [Tyr, 50µM] (B) in the presence of DMSO or N 6 -MB-cAMP [500µM and 1mM] for 20h. Neutrophils isolated from COPD patients were incubated with DMSO or tyrphostin AG825 [50µM] in the presence of DMSO or N 6 -MB-cAMP [500µM] for 20h (C). Neutrophils isolated from COPD patients and age-matched healthy control subjects (HC) were incubated with DMSO, erbstatin (D) [20, 40µM] or tyrphostin AG825 (E) [25, 50µM] in the presence or absence of GMCSF [50u/mL] for 20h. Apoptosis was assessed by light microscopy. The data are expressed as mean percentage apoptosis ± SEM from 4-6 independent experiments. Statistical significances were calculated by one-way ANOVA with appropriate post-test and indicated as *p<0.05, **p<0.01, ***p<0.001. (F) Neutrophils were incubated with DMSO or tyrphostin AG825 [Tyr, 50µM] for 60 min before the addition of GMCSF [50u/mL] for 15 or 30 mins. (G) Neutrophils were incubated with DMSO, tyrphostin AG825 [50µM] for 60 min before the addition of GMCSF [50u/mL] for a further 7h. Cells were lysed, subjected to SDS-PAGE electrophoresis and membranes probed for p-AKT, Mcl-1 or loading controls, AKT and P38. Images are representative of 3 independent experiments. Charts show densitometric values of 3 individual immunoblots and are expressed as a ratio of target (p-AKT or Mcl-1) over loading control (AKT or P38, respectively).

Article Snippet: Membranes were then blocked with 5% skimmed milk in TBS-tween and probed against antibodies to p-AKT (Cell Signalling Technology), AKT (Cell signalling Technology), Mcl-1 (Santa Cruz Biotechnology) or p38 (loading control, StressMarq Biosciences Inc.), followed by HRP-conjugated secondary antibodies and detection with chemiluminescent substrate solution ECL2 (GE Healthcare).

Techniques: Incubation, Isolation, Light Microscopy, SDS Page, Electrophoresis, Western Blot

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: The effects of melatonin on oxidative stress, inflammation, apoptosis and Nrf2/HO-1 in acrylamide-induced lung injury in rats

doi: 10.1007/s00210-025-04292-8

Figure Lengend Snippet: Lung TNF-α, IL-1β, IL-6, NF-κB, iNOS, COX-2, MPO, p38 MAPK, and IL-10 activities in experimental groups ( n = 10). Results are expressed as mean ± SEM

Article Snippet: Catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH), heme oxygenase 1 (HO-1), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), malondialdehyde (MDA), nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear factor kappa (NF-κB), superoxide dismutase (SOD), tumor necrosis factor alpha (TNF-α), caspase 3 (CASP3), inducible nitric oxide synthase (iNOS; NOS2), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinases (p38-MAPK), and myeloperoxidase (MPO) enzyme-linked immunosorbent assay (ELISA) kits were purchased from BT-LAB.

Techniques:

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 2. Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 M). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 3. Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-B activation. A, HEK293 cells were left untreated, or were trans- fected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simul- taneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si- Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8- reporter (B) or NF-B reporter construct (C and D) and a -galactosi- dase reporter plasmid. Cells were either left untreated () or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were tran- siently transfected with a control vector (ctrl) or NOD2 expression plas- mid along with an IL-8 luciferase reporter plasmid (E) or a NF-B- driven luciferase reporter (F), respectively, and a -galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild- type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively ac- tive Rac1L61. Cells were either stimulated with 10 g/ml MDP (MDP) or left untreated (), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-B-driven luciferase reporter and a -galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or con- stitutive active Rac1 (Rac1L61) mean SD; , p 0.01.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Activation Assay, Control, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Luciferase, Dominant Negative Mutation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 6. Involvement of -PIX in NOD2-medi- ated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of -Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting -Pix (si--Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti--Pix Abs were performed. Western blots were si- multaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 g/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti- NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data pre- sented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Sequencing, Western Blot, Incubation, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Computational and In Vitro Analysis of Plumbagin’s Molecular Mechanism for the Treatment of Hepatocellular Carcinoma

doi: 10.3389/fphar.2021.594833

Figure Lengend Snippet: (A) Protein expression levels of LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK in SMMC-7721 cells measured by Western blot after treatment with 5 μM PL, 10 μM 3-MA autophagy inhibitor, 10 μM rapamycin autophagy agonist, 10 μM SB202190, and 10 μM SB203580 p38 MAPK inhibitor. (B) LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK were used as internal reference total proteins for gray value analysis. * p < 0.05, ** p < 0.01 *** p < 0.001, compared with the PL group. # p < 0.05, ## p < 0.01 ### p < 0.001 vs. the control group. (C) Protein expression levels of LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK in BEL-7404 cells measured by Western blot after treatment with 5 μM PL, 10 μM 3-MA autophagy inhibitor, 10 μM rapamycin autophagy agonist, 10 μM SB202190, and 10 μM SB203580 p38 MAPK inhibitor. (D) LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK were used as internal reference total proteins for gray value analysis. * p < 0.05, ** p < 0.01 *** p < 0.001, compared with the PL group. # p < 0.05, ## p < 0.01 ### p < 0.001 vs. the control group.

Article Snippet: Plumbagin (PL) was purchased from Sigma-Aldrich (St. Louis, MO, United States) with purity ≥98%; the Acridine Orange (AO)/Ethidium bromide (EB) Double Stain Kit was from Solable Technology (Beijing, China); N-acetyl-l-cysteine, SB203580, and SB202190 were from Sigma-Aldrich (St. Louis, MO, United States); SC-79, MEK2206, 3-MA, and Z-VAD-FMK were from Selleck (Texas, United States); the BCA Protein Assay Kit, ROS Assay Kit, Annexin V-FITC Apoptosis Detection Kit and Cell lysis buffer for Western were all obtained from Beyotime Biotechnology (Shanghai, China); antibodies against Akt, phospho-Akt, mTOR, phospho-mTOR, p38 MAPK, phospho-p38 MAPK, PI3K, phospho-PI3K, LC3B, cleave-RP, and cleave-caspase 3 were from Cell Signaling Technology, Inc. (Boston, MA, United States); and β-p38 MAPK was purchased from Boster Technology (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: Science Advances

Article Title: A mouse model of 22q11.2 deletions: Molecular and behavioral signatures of Parkinson’s disease and schizophrenia

doi: 10.1126/sciadv.aar6637

Figure Lengend Snippet: ( A and B ) The ACC, CPu, and SNc in Df1 /+ mice ( n = 4) and the WT littermates ( n = 4) (3.5 months old) were immunostained with anti–α-synuclein (α-Sync) (A) or anti-p62 (B) antibodies, together with anti-CaMKII, anti–DARPP-32, and anti-TH antibodies, which label the most abundant neuronal cell type in the ACC, CPu, and SNc, respectively. Images were acquired by a confocal microscope using the same parameters across multiple specimens. Images in the ACC and SNc are shown. Scale bars, 20 μm. Relative fluorescence intensities (arbitrary units) of α-synuclein or p62 immunostaining were measured from each soma colabeled with the neuronal marker (30 to 50 soma per section) and plotted in the graph. Overall average fluorescence intensities (±SEM) calculated across three to four serial sections per animal from all animals used ( n = 4 per group) are shown in red in the graph. * P < 0.05 and ** P < 0.01 (two-tailed Mann-Whitney test).

Article Snippet: The brains were then serially cut into 50-μm-thick coronal sections using a vibratome (VT1200 S, Leica), and the sections from one cohort of mice (a littermate pair of WT and Df1 /+ mice) were simultaneously immunostained for 16 hours at 4°C with primary antibodies anti-p62 (guinea pig, 1:400; Medical and Biological Laboratories), anti–α-synuclein (rabbit, 1:200; Immuno-Biological Laboratories), anti-CaMKII (mouse, 1:1000; StressMarq), anti–DARPP-32 (mouse, 1:1000; Calbiochem), and anti-TH (mouse, 1:3000; Abcam), followed by incubation with Alexa Fluor 488– or Alexa Fluor 546–conjugated secondary antibodies (1:500; Gibco).

Techniques: Microscopy, Fluorescence, Immunostaining, Marker, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: p38MAPK inhibition by SB203580 increases αSyn turnover and lowers αSyn secretion and toxicity in cells expressing p25α. A , NGF-differentiated PC12-αSyn/p25α cells were treated with SB203580 as indicated with doxycycline (to induce αSyn and p25α transgenes) for 2 days before Western blot analysis of αSyn in TCA-precipitated conditioned medium and actin and αSyn in the lysate fraction (ordinary one-way ANOVA, N = 4–6). B , PC12-αSyn/p25α cell cultures received SB203580 either concurrently with doxycycline treatment (concurrently) or 24 h after doxycycline induction (post). After 2 days, conditioned medium was analyzed for lactate dehydrogenase (LDH) to assess cell death (Kruskal–Wallis test, N = 4). C , αSyn and p25α expression was induced in PC12-αSyn/p25α cells by doxycycline treatment for 24 h with/without concurrent SB203580 administration followed by doxycycline washout. Cells were chased for a further 4 days with/without SB203580 with sampling of replicate wells each day for analysis of intracellular αSyn by Western blotting. The Western blot shows a representative experiment, whereas graphs represent αSyn normalized to actin Western blot band absorbance relative to D1 values (two-way ANOVA, N = 3). D , NGF-differentiated PC12 cells expressing αSyn or αSyn/p25α were treated or not with SB203580 at 1 or 2 μM for 2 days of transgene expression and then lysates were analyzed by Western blotting for total and phosphorylated p38MAPK and actin. The graphs show mean integrated absorbance of Western blot bands for the ( D ) ratio of phosphorylated to total p38MAPK ( i.e. , specific activity) for a SB203580 concentration of 2 μM, the ratio being derived from the absorbances of ( E ) total p38MAPK and ( F ) p-p38MAPK bands (Kruskal–Wallis test, N = 4). All graphs show mean ± SEM. αSyn, α-synuclein; NGF, nerve growth factor; TCA, trichloroacetic acid.

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: Inhibition, Expressing, Western Blot, Sampling, Activity Assay, Concentration Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: shRNA knockdown of p38MAPK-α reproduces the effects of SB203580 treatment. A , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α (one shRNA) and p38MAPK-γ isoforms (two shRNAs), incubated with doxycycline for 2 days, and subsequently lysed and Western blotted for expression of p38MAPK isoforms (using anti-p38MAPKα antibody 9212 from Cell Signaling) or phosphorylated p-p38MAPK. Cropped lanes are derived from the same membrane. B , the same cells were analyzed by Western blotting for expression of αSyn in the conditioned medium, and αSyn, LC3, and p62 in the lysate. C – E , quantitation of Western blot band absorbances from aforementioned experiment for double p38MAPK-α and p38MAPK-γ knockdown for ( C ) αSyn in conditioned medium, or ( D ) αSyn, and ( E ) LC3-II in the lysate fraction. (Kruskal–Wallis test, N = 4–6). F , LDH release from the same cells (ordinary one-way ANOVA, N = 4). G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used. Cropped lanes are derived from the same membrane. H and I , the graphs show quantitation of levels of p38MAPK-α isoform (unpaired t test, N = 4) and active (phosphorylated) p38MAPK (one-sample t test, N = 4). J , secretion of αSyn to the medium (unpaired t test, N = 3) as performed in ( G ). K , LDH release to the medium (unpaired t test, N = 4). All graphs show mean ± SEM. αSyn, α-synuclein; LDH, lactate dehydrogenase.

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: shRNA, Knockdown, Transduction, Expressing, Incubation, Western Blot, Derivative Assay, Membrane, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: SB203580 decreases p-ser129 αSyn staining in primary neurons. A , primary mouse cortical neurons expressing transgene human αSyn were treated with 1 μg/ml αSyn fibrils ±1 or 5 μM SB203580 at DIV5 in the further presence or not of lysosomal inhibitors leupeptin, pepstatin, and E64 (LPE) and harvested DIV7. B and C , neuronal cultures were fixed and stained with primary antibodies for ( B ) p-Ser129 αSyn and ( C ) Hoechst (to assess live nuclei) for quantitation by Cellomics Array Scanner analysis (ordinary one-way ANOVA, N = 3). D , representative spinning disc confocal microscopy images of neuronal cultures stained for p-Ser129 ( green ), Map2 ( red ), and Hoechst ( gray ) from a Cellomics experiment as aforementioned; bars represent 32 μm. E , cell lysates prepared from DIV7 neuronal cultures, treated with SB203580 and/or LPE in the absence of αSyn fibrils as indicated, were Western blotted with anti-LC3, anti-p62, anti-p-p38MAPK, or anti-actin antibodies as shown. F – H , quantitation of aforementioned immunoblots was performed for ( F ) LC3-II, ( G ) p62/SQSTM1, and ( H ) p-p38MAPK (without αSyn fibrils) (ordinary one-way ANOVA for F and G and Kruskal–Wallis test for H , N = 3). All graphs show mean ± SEM. αSyn, α-synuclein; DIV, days in vitro .

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: Staining, Expressing, Quantitation Assay, Confocal Microscopy, Western Blot, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: αSyn degradation under basal conditions and p38MAPK inhibition. Under basal conditions αSyn is degraded by multiple mechanisms first and foremost by macroautophagy and Ndfip1-mediated internalization of αSyn; however, chaperone-mediated autophagy (LAMP2a and Hsc70) also contributes. When p25α-mediated p38MAPK activation is opposed pharmacologically or genetically, the majority of αSyn is turned over in an ESCRT-dependent process relying on Hsc70 and TSG101, and αSyn degradation commences in late endosomes. Under basal conditions, lysosome fusion with autophagosomes and amphisomes (the fusion product of an autophagosome with a late endosome) is partially blocked by p25α, which results in their exocytosis and release of αSyn. In contrast, under conditions of p38MAPK inhibition, ESCRT-dependent αSyn import and degradation begins in the late endosome, and the endosomal pathway runs to completion to deliver αSyn to lysosomes for degradation. αSyn, α-synuclein; ESCRT, endosomal sorting complex required for transport; LAMP, lysosome-associated membrane protein.

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: Inhibition, Activation Assay, Membrane