phospho p38α thr180 tyr182  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: Targeting TTK Inhibits Tumorigenesis of T‐Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway

doi: 10.1002/advs.202413990

Figure Lengend Snippet: TTK interacted with p38α through C‐terminal and phosphorylated p38α in TCL. A) GO enrichment analysis of differentially phosphorylated proteins. The circle area indicated the number of genes, while the circle color represented the range of the corrected P values. B) 12 potential kinase substrates of TTK were identified by integrating the results of motif prediction percentile and phosphoproteomics quantification. Bar plot showed the fold change of differentially phosphorylated proteins. C) The predicted binding mode of TTK (blue) and p38α (green). D) Co‐IP assay showed the interaction between TTK and p38α in TCL. E) Immunofluorescence co‐staining of TTK and p38α in TCL. Bar = 20 µm. F) Immunofluorescence distribution showed the co‐localization of TTK (red) and p38α (green). G) The schematic of TTK truncated fragments. H) Co‐IP assay confirmed the interaction between TTK 525–857aa and p38α in TCL. I) WB analysis showed the expression level of p38α and p‐p38α after TTK knockdown in TCL. J) WB analysis showed the expression level of p38α and p‐p38α after CFI‐402257 treatment (10 µM, 48 h) in TCL. K) WB analysis showed the expression level of p38α and p‐p38α after transfected with TTK kinase inactivated mutation plasmid. L‐M) CCK8 assay showed that the proliferation of TCL cells was inhibited by TTK kinase inactivation and p38 phosphorylation inactivation plasmids, which was reversed by p38 phosphorylation activation plasmid (n = 3). N) Detection of cell apoptosis in TCL cells after TTK knockdown and p38α phosphorylation activation plasmid transfection by flow cytometry (n = 3). O) Detection of cell cycle in TCL cells after TTK knockdown and p38α phosphorylation activation plasmid transfection by flow cytometry (n = 3). Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies were as follows, TTK (A2500, Abclonal, Wuhan, China), p38α (66234‐1‐Ig, Proteintech), Caspase‐3 (ab184787, Abcam, MA, USA), cleaved‐Capase‐3 (68773‐1‐Ig, Proteintech), Caspase‐9 (10380‐1‐AP, Proteintech), cleaved‐Caspase‐9 (9509, Cell Signaling Technology (CST), MA, USA), PARP (13371‐1‐AP, Proteintech), LC3B (ab192890, Abcam), phospho‐p38α (Thr180/Tyr182) (4511, CST), AKT (60203‐2‐Ig, Proteintech), phospho‐AKT (2535, CST), AMPK (ab32047, Abcam), phospho‐AMPK (2535, CST), mTOR (2983, CST), phospho‐mTOR (5536, CST), GAPDH (10381‐1‐AP, Proteintech), p62 (ab207305, Abcam), Beclin (ab207612, Abcam), c‐myc (18583, CST), p‐H2AX (9718, CST), Ubiquitin (3936, CST), HA (3724, CST), TSC2 (24601‐1‐AP, Proteintech), p‐TSC2 (5584, CST), Raptor (20984‐1‐AP, Proteintech), phospho‐Raptor (2083, CST).

Techniques: Phospho-proteomics, Binding Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Knockdown, Transfection, Mutagenesis, Plasmid Preparation, CCK-8 Assay, Activation Assay, Flow Cytometry

Journal: Advanced Science

Article Title: Targeting TTK Inhibits Tumorigenesis of T‐Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway

doi: 10.1002/advs.202413990

Figure Lengend Snippet: Inhibiting TTK activated the AMPK/mTOR pathway through p38α to restrain TCL development. A) KEGG enrichment analysis of differentially phosphorylated proteins. The circle area indicates the number of genes in the pathway, while the circle color represents the range of the corrected P values. B,C) WB analysis showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown and CFI‐402257 treatment (10 µM, 48 h). D) WB analysis showed the expression level of p‐AMPK and p‐mTOR after transfection with p38α phosphorylation inactivation plasmid in TCL. E) WB analysis showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown and transfection with p38α phosphorylation activation plasmid. F) CCK8 assay showed that the combined treatment with Rapamycin (mTOR inhibitor, 10 µM, 48 h) enhanced anti‐tumor effects of CFI‐402257 (10 µM, 48 h) in TCL (n = 3). G) WB analysis showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown and Dorsomorphin (AMPK inhibitor) treatment (10 µM, 48 h). H) CCK8 assay showed that Dorsomorphin (AMPK inhibitor, 10 µM, 48 h) and MHY1485 (mTOR activator, 20 µM, 48 h) rescued the inhibitory effect of TTK knockdown on TCL cell proliferation (n = 3). I) Mechanism diagram summarized that TTK contributed to TCL development through regulating the p38α/AMPK/mTOR axis. Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies were as follows, TTK (A2500, Abclonal, Wuhan, China), p38α (66234‐1‐Ig, Proteintech), Caspase‐3 (ab184787, Abcam, MA, USA), cleaved‐Capase‐3 (68773‐1‐Ig, Proteintech), Caspase‐9 (10380‐1‐AP, Proteintech), cleaved‐Caspase‐9 (9509, Cell Signaling Technology (CST), MA, USA), PARP (13371‐1‐AP, Proteintech), LC3B (ab192890, Abcam), phospho‐p38α (Thr180/Tyr182) (4511, CST), AKT (60203‐2‐Ig, Proteintech), phospho‐AKT (2535, CST), AMPK (ab32047, Abcam), phospho‐AMPK (2535, CST), mTOR (2983, CST), phospho‐mTOR (5536, CST), GAPDH (10381‐1‐AP, Proteintech), p62 (ab207305, Abcam), Beclin (ab207612, Abcam), c‐myc (18583, CST), p‐H2AX (9718, CST), Ubiquitin (3936, CST), HA (3724, CST), TSC2 (24601‐1‐AP, Proteintech), p‐TSC2 (5584, CST), Raptor (20984‐1‐AP, Proteintech), phospho‐Raptor (2083, CST).

Techniques: Expressing, Knockdown, Transfection, Phospho-proteomics, Plasmid Preparation, Activation Assay, CCK-8 Assay

Journal: Advanced Science

Article Title: Targeting TTK Inhibits Tumorigenesis of T‐Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway

doi: 10.1002/advs.202413990

Figure Lengend Snippet: TTK regulated the autophagy in TCL via modulating p38α phosphorylation. A) IF analysis of LC3B autophagic vesicles (red) after CFI‐402257 treatment (10, 20µM, 48 h) in TCL cells. Bar = 20 µm. B) IF analysis of LC3B autophagic vesicles (red) after TTK knockdown in TCL cells. Bar = 20 µm. C) IF analysis of LC3B autophagic vesicles (red) after transfection with p38α phosphorylation inactivation plasmid in TCL. Bar = 20 µm. D) WB analysis showed the expression level of p62, Beclin and LC3B‐II after CFI‐402257 treatment (10 µM, 48 h) in TCL cells. E) WB analysis showed the expression level of p62, Beclin, and LC3B‐II after TTK knockdown in TCL. F) WB was used to determine the expression level of p62, Beclin, and LC3B‐II after TTK knockdown and chloroquine treatment (10µM, 12 h) in TCL. G) WB analysis showed the expression level of p62, Beclin, and LC3B‐II after transfection with p38α phosphorylation inactivation plasmid in TCL. H) WB analysis showed the expression level of p62, Beclin and LC3B‐II after TTK knockdown and transfection with p38α phosphorylation activation plasmid in TCL. Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies were as follows, TTK (A2500, Abclonal, Wuhan, China), p38α (66234‐1‐Ig, Proteintech), Caspase‐3 (ab184787, Abcam, MA, USA), cleaved‐Capase‐3 (68773‐1‐Ig, Proteintech), Caspase‐9 (10380‐1‐AP, Proteintech), cleaved‐Caspase‐9 (9509, Cell Signaling Technology (CST), MA, USA), PARP (13371‐1‐AP, Proteintech), LC3B (ab192890, Abcam), phospho‐p38α (Thr180/Tyr182) (4511, CST), AKT (60203‐2‐Ig, Proteintech), phospho‐AKT (2535, CST), AMPK (ab32047, Abcam), phospho‐AMPK (2535, CST), mTOR (2983, CST), phospho‐mTOR (5536, CST), GAPDH (10381‐1‐AP, Proteintech), p62 (ab207305, Abcam), Beclin (ab207612, Abcam), c‐myc (18583, CST), p‐H2AX (9718, CST), Ubiquitin (3936, CST), HA (3724, CST), TSC2 (24601‐1‐AP, Proteintech), p‐TSC2 (5584, CST), Raptor (20984‐1‐AP, Proteintech), phospho‐Raptor (2083, CST).

Techniques: Phospho-proteomics, Knockdown, Transfection, Plasmid Preparation, Expressing, Activation Assay

Journal: Pharmaceutics

Article Title: Improving Water Solubility and Skin Penetration of Ursolic Acid through a Nanofiber Process to Achieve Better In Vitro Anti-Breast Cancer Activity

doi: 10.3390/pharmaceutics16091147

Figure Lengend Snippet: Ursolic acid (UA) and its nanofiber (UANF) affected the protein expression related to anti-breast cancer in MCF-7 cells. ( a ) Phosphorylation of p38 and ERK1/2, and the protein expression of ( b ) STAT3, cleaved caspase-3, and ( c ) TNF-α. # p < 0.05 significance with negative control. * p < 0.05 significance with UA/PBS.

Article Snippet: Primary antibodies included phospho-signal transducer and activator of transcription 3 (STAT3) (Cell Signaling Technology, Danvers, MA, USA), GAPDH, p38, and extracellular regulated protein kinases (ERK) (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-p38α and phospho-ERK (Merck Millipore, Burlington, MA, USA), tumor necrosis factor (TNF)-α and cleaved caspase-3 (ABclonal Technology, Woburn, MA, USA).

Techniques: Expressing, Phospho-proteomics, Negative Control

Journal: Molecular Autism

Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

doi: 10.1186/s13229-024-00595-4

Figure Lengend Snippet: Phosphorylated p38 level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were stained with anti-p38α (1:800, AF8691, R&D Systems), anti-p38α (1:800, 4511, cell Signaling Technology, Inc.), and anti-AgRP (1:500, PA5-47831, Invitrogen) antibodies overnight at 4 °C, and then with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) labeled secondary antibodies (donkey anti-rabbit or donkey anti-goat) for 1 h at room temperature.

Techniques: Knockdown, Protein-Protein interactions, Western Blot, Phospho-proteomics, Two Tailed Test, Software

Journal: Molecular Autism

Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

doi: 10.1186/s13229-024-00595-4

Figure Lengend Snippet: p38α overexpression in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm), p38α (green, 488 nm, FITC), and Neun (red, 561 nm, TRITC) in ARC. B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox and AAV9-Agrp-Cre mix injection to ARC in WT mice, green fluorescent protein (GFP, 488 nm) represents virus expression. C and D . Groom time and bouts in 24 h of p38α ARC−con and p38α ARC−OE mice. E . The first phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger2 chamber. G . Total distance in 24 h of p38α ARC−con and p38α ARC−OE mice. H . Activity time in 24 h of p38α ARC−con and p38α ARC−OE mice. 10-week-old WT male mice were injected with virus. The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α ARC−con group, 3 mice were excluded and 9 mice were included in the experiment. 11 injected mice in the p38α ARC−OE group, 3 mice were excluded and 8 mice were included in the experiment. Home-Cage monitoring test was performed 6 weeks after virus injection, three-chamber test was performed 8 weeks after virus injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

Article Snippet: Sections were stained with anti-p38α (1:800, AF8691, R&D Systems), anti-p38α (1:800, 4511, cell Signaling Technology, Inc.), and anti-AgRP (1:500, PA5-47831, Invitrogen) antibodies overnight at 4 °C, and then with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) labeled secondary antibodies (donkey anti-rabbit or donkey anti-goat) for 1 h at room temperature.

Techniques: Over Expression, Immunofluorescence, Staining, Injection, Virus, Expressing, Activity Assay, Two Tailed Test, Software

Journal: Molecular Autism

Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

doi: 10.1186/s13229-024-00595-4

Figure Lengend Snippet: Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm) and p38α (green, 488 nm, FITC) in ARC, AgRP-tomato mice were generated by mating Agrp-Cre mouse with Tomato-reporter mouse (red, 561 nm). B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox stereotaxic injection to ARC in Agrp-Cre mice, GFP represents virus expression. C and D , Groom time and bouts in 24 h of p38α AgRP−con and p38α AgRP−OE mice. E . The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber. G . The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice. H . The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice. 10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox . The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment. 12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment. Home-Cage monitoring test was performed 4–6 weeks after AAV injection, three-chamber test was performed 8–10 weeks after AAV injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

Article Snippet: Sections were stained with anti-p38α (1:800, AF8691, R&D Systems), anti-p38α (1:800, 4511, cell Signaling Technology, Inc.), and anti-AgRP (1:500, PA5-47831, Invitrogen) antibodies overnight at 4 °C, and then with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) labeled secondary antibodies (donkey anti-rabbit or donkey anti-goat) for 1 h at room temperature.

Techniques: Over Expression, Immunofluorescence, Staining, Generated, Injection, Virus, Expressing, Activity Assay, Control, Two Tailed Test, Software

Journal: Molecular Autism

Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

doi: 10.1186/s13229-024-00595-4

Figure Lengend Snippet: Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . schematic of generating p38α AgRP-176/327 mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. D . the total distance in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. E . the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. F . the first phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber. p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group. Mice were subjected to Home-Cage monitoring test at the age of 8–9 weeks, and subjected to three-chamber test at the age of 9–10 weeks. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

Article Snippet: Sections were stained with anti-p38α (1:800, AF8691, R&D Systems), anti-p38α (1:800, 4511, cell Signaling Technology, Inc.), and anti-AgRP (1:500, PA5-47831, Invitrogen) antibodies overnight at 4 °C, and then with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) labeled secondary antibodies (donkey anti-rabbit or donkey anti-goat) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Activity Assay, Two Tailed Test, Software

Journal: Molecular Autism

Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

doi: 10.1186/s13229-024-00595-4

Figure Lengend Snippet: Inactivated p38α in AgRP neurons ameliorates the autistic-like behaviors of Shank3 -/- mice. A . scheme of generating Shank3 -/- : p38α AgRP-180/182 mice and immunofluorescence staining of p-p38α(green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. D . the total distance in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. E . the activity time in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. F . the first phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger2 chamber. Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 male mice were subjected to Home-Cage monitoring test at the age of 8 weeks, and subjected to three-chamber test at the age of 14 weeks, 12 mice in Shank3 -/- : p38α AgRP-con and 19 mice in Shank3 -/- : p38α AgRP-180/182 group. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

Article Snippet: Sections were stained with anti-p38α (1:800, AF8691, R&D Systems), anti-p38α (1:800, 4511, cell Signaling Technology, Inc.), and anti-AgRP (1:500, PA5-47831, Invitrogen) antibodies overnight at 4 °C, and then with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) labeled secondary antibodies (donkey anti-rabbit or donkey anti-goat) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Activity Assay, Two Tailed Test, Software

Journal: Cell Death & Disease

Article Title: Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control

doi: 10.1038/s41419-024-06423-0

Figure Lengend Snippet: A , B Various cancer cell lines were treated with DMSO (control) or SB203580 (50 µM) for 2–24 h and assessed for vacuolation and cell death by phase-contrast microscopy and flow cytometry (annexin V-propidium iodide staining), respectively (†, not determined; see also Supplementary Fig. ). At 24 h, <3% of DMSO-treated cells were vacuolated and cell death values (%) were: DU145 (9 ± 0.4), A549 (12.8 ± 0.8), HCT116 (15.0 ± 1.6), HT-29 (10.2 ± 1.2). The number of vacuoles per cell and the average vacuole size (µm 2 ) was determined using ImageJ analysis of phase-contrast images. C DU145 cells were stained with CFDA-SE, exposed to DMSO (shaded peaks) or SB203580 (empty peaks), and monitored for changes in cell proliferation, as described in the methods. D DU145 cells were treated with SB203580 (50 µM) for 24 h and then washed and replaced with fresh media ± SB203580 for 4–24 h. At each time point following the washout, cells were examined for vacuolation by phase-contrast microscopy (see also Supplementary Fig. ). E DU145 prostate cancer cells were treated with increasing concentrations of the pharmacological p38 MAPK inhibitor, SB203580 (0–100 µM), for 24 h and examined for signs of vacuolation by phase-contrast microscopy (200×). Inset: SB203580 (50 µM) inhibited p38-dependent sequential phosphorylation of MK2 and HSP27. Concentration-dependent inhibition of HSP27 phosphorylation by SB203580 (0–100 µM) was also determined by western blotting (see Supplementary Fig. ) with individual bands scanned, quantified with ImageJ software, and plotted as the percent of p-HSP27 inhibited. F , G DU145 cells were transiently transfected with expression plasmids encoding EGFP, constitutively active (D176A/F327S) p38α (EGFP-p38α-CA), or p38α-CA containing an additional mutation to the gatekeeper residue (T106M) that renders p38α resistant to SB203580 [EGFP-p38α-CA (T106M)]. EGFP-positive cells were then evaluated by fluorescence microscopy for the number of vacuoles present per cell. H , I p38α and p38β were deleted from DU145 cells using CRISPR-Cas9; and three p38 DKO clones were exposed to SB203580 (50 µM) or SB202190 (50 µM) and evaluated for vacuolation by phase-contrast microscopy using ImageJ analysis software.

Article Snippet: Antibodies to ATG5 (#2630), ATG12 (#4180), ATG16L1 (#8089), Beclin 1 (#3738), β-actin (#4970), CTSD (#2284), LC3B (#2775), p62 (#5114), p38α (#9217), p38β (#2339), phospho-p38α (pThr-180/pThr-182; #9216), phospho-hsp27 (pSer-82; #2406), phospho-MK2 (pThr-222; #3316), Rab7 (#95746), and Rab9 (#5118) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Microscopy, Flow Cytometry, Staining, Phospho-proteomics, Concentration Assay, Inhibition, Western Blot, Software, Transfection, Expressing, Mutagenesis, Residue, Fluorescence, CRISPR, Clone Assay

Journal: Cell Death & Disease

Article Title: Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control

doi: 10.1038/s41419-024-06423-0

Figure Lengend Snippet: A Model to explain how SB203580 and SB202190 induce vacuolation through combined inhibition of p38 MAPKs and PIKfyve. B , C p38 DKO clones were exposed to YM201636 (500 nM) or apilimod (20 nM) for 24 h and evaluated for vacuolation by phase-contrast microscopy using ImageJ analysis software. D , E p38 DKO cells were stably reconstituted with a vector control, or wild-type (WT), constitutively active (CA), or kinase dead (KD) versions of p38α, exposed to apilimod (20 nM), and evaluated for vacuolation by phase-contrast microscopy using ImageJ analysis software. F Model for epistatic relationship between PIKfyve and p38 MAPKs. G – I Wild-type DU145 cells were treated with apilimod (50 nM) for 24 h, after which apilimod was removed and BIRB-796 (50 µM) was added to selectively inhibit p38 MAPKs during the “washout” phase. Vacuolation was assessed by phase-contrast microscopy over time (0–12 h) using ImageJ software to determine both the number of vacuoles per cell and percent cell area vacuolated. Inhibition of p38 MAPKs delays the resolution of vacuoles, implicating a role for p38 MAPKs in promoting LEL fission.

Article Snippet: Antibodies to ATG5 (#2630), ATG12 (#4180), ATG16L1 (#8089), Beclin 1 (#3738), β-actin (#4970), CTSD (#2284), LC3B (#2775), p62 (#5114), p38α (#9217), p38β (#2339), phospho-p38α (pThr-180/pThr-182; #9216), phospho-hsp27 (pSer-82; #2406), phospho-MK2 (pThr-222; #3316), Rab7 (#95746), and Rab9 (#5118) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Clone Assay, Microscopy, Software, Stable Transfection, Plasmid Preparation, Control

Journal: Molecular neurobiology

Article Title: Nimbolide Targets Multiple Signalling Pathways to Reduce Neuroinflammation in BV-2 Microglia.

doi: 10.1007/s12035-023-03410-y

Figure Lengend Snippet: Fig. 8 Nimbolide targets MAPK activation in BV-2 microglia. Cells were treated with nimbolide (125, 250 and 500 nM) prior to stimulation with LPS (100 ng/ml). Immuno- blot analyses shows reduction in protein expression of phospho- p38 (A, B), and phospho-JNK (C, D). Densitometric values are expressed as mean ± SEM for at least three independent experiments. Statistical analysis was performed using one-way ANOVA with post hoc Tukey test (multiple comparisons). ns (not significant) ***p<0.001, ****p<0.0001 versus LPS control

Article Snippet: Primary antibodies used were rabbit anti-iNOS (Cell signalling, 1:1000), rabbit anti-COX-2 (Cell signalling, 1:1000), rabbit anti-phospho-p65 (Cell signalling, 1:1000), κ rabbit anti-phospho-IκBα (Abcam, 1:5000), rabbit antiphospho-JNK (Cell signalling, 1:1000), rabbit anti-totalJNK (Cell signalling, 1:1000), rabbit anti-phospho-p38α (Cell signalling, 1:1000), rabbit anti-total-p38 (Santa Cruz, 1:500), rabbit anti-gp91phox (Abcam, 1:5000), rabbit anti-Keap1 (Santa Cruz, 1:500), mouse antiNrf2 (Santa Cruz, 1:500), rabbit anti-HO1 (Santa Cruz, 1:500), rabbit anti-NQO1 (Santa Cruz, 1:500), rabbit anti-acetyl-p65 (Cell Signalling, 1:1000), rabbit antiSIRT1 (Santa Cruz, 1:500), rabbit anti-actin (Sigma, 1:1000) and rabbit anti-lamin B1 (Santa Cruz, 1:500).

Techniques: Activation Assay, Expressing, Control