Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 1. Phospho-proteomic analysis of p38- and JNK-dependent phosphorylation reactions. (a). Schematic of phospho- proteomic workflow. U2OS cells were treated in duplicates with indicated combinations of anisomycin (Ani, 1 h) and p38 and JNK inhibitors (p38i, JNKi, 0.5 h pre-treatment). Lysates were digested with trypsin and peptides were labeled with

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Phospho-proteomics, Labeling

Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 3. GIGYF1 phospho-mutant retains p-body localization upon cellular stress. (a). U2OS cells were transfected with the indicated siRNA, pre-treated with p38 and MK2 inhibitors (0.5 h) and UV-irradiated (50 J/m2, 1 h recovery) as indicated. Lysates were incubated with recombinant GST-14–3–3 protein or GST alone. GST pull-down material (PD: GST) and whole

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Mutagenesis, Transfection, Irradiation, Incubation, Recombinant

Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 4. JNK phosphorylation targets in the Golgi apparatus. (a). JNK- and p38-dependent phosphorylation sites on Golgi apparatus-resident proteins or Golgi trafficking proteins extracted from Figure 1c, Table S1 and [11]. (b). U2OS cells were pre-treated with JNK and p38 inhibitors (JNKi, p38i, 0.5 h) and treated with anisomycin (Ani, 1 h) as indicated. Lysates were separated by SDS-PAGE or phos-tag gel and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Phospho-proteomics, SDS Page, Western Blot

Journal: International journal of molecular sciences

Article Title: Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

doi: 10.3390/ijms22179595

Figure Lengend Snippet: Figure 5. Regulation of Golgi morphology by p38 and JNK. (a). Heatmap and horizontal clustering of descriptors of Golgi morphology. U2OS cells were pre-treated with the combination of JNK and p38 inhibitors (JNKi, p38i, 0.5 h) and treated with anisomycin (Ani, 1 h) or IL1β (1 h) as indicated. Cells were fixed, immunostained with antibodies against cis Golgi markers GM130, GRASP65 and/or trans Golgi markers GRASP55 and TGN46 and images were acquired by high content microscopy. Images were processed and analyzed with CellProfiler software for calculation of the indicated parameters, and are presented as log2-transformed mean fold changes compared to the control. n > 1700 cells. (b). Box plots of selected

Article Snippet: Membranes were blocked in PBS-T + 5% milk and then probed with the following antibodies overnight: Phospho RxxS*/T* (Cell signaling, 9614, rabbit), GIGYF1 (Abcam, ab121784 and ab121646, rabbit), MK2 (Cell signaling, 3042, rabbit), CHK1 (S345, Cell signaling, 2358, rabbit), 14–3–3 (pan, Cell signaling, 8312, rabbit), GST (Santa Cruz, discontinued, sc459, rabbit), TTP (Sigma, T5327, rabbit), ZNF598 (Abcam, ab111698, rabbit and/or Sigma, HPA041760, rabbit), FLAG (Sigma, F1804, rabbit and/or Sigma, F1804, mouse) or 4EHP (Cell signaling, 6916, rabbit), GRASP55 (Abcam, Ab211532, mouse), CAMSAP2 (Novus, Abingdon, UK, NCP1–21402, rabbit), TJAP1 (Sigma, HPA030165, rabbit), GM130 (BD biosciences, Franklin Lakes, NJ, USA Clone 35, mouse), YIPF2 (Santa Cruz, sc-398530, mouse), p-JNK (Cell signaling, 9255, mouse), p-p38 (T180/Y182, Cell signaling, 9216, mouse), p150 (BD biosciences, 610473, mouse), MCM6 (Santa Cruz, goat), HA (Santa Cruz, sc-7392, mouse) and GFP (Torrey Pines, Secaucus, NJ, USA TP401, rabbit).

Techniques: Microscopy, Software, Transformation Assay, Control

Journal: Frontiers in oncology

Article Title: CDKN2A Deletion in Melanoma Excludes T Cell Infiltration by Repressing Chemokine Expression in a Cell Cycle-Dependent Manner.

doi: 10.3389/fonc.2021.641077

Figure Lengend Snippet: FIGURE 3 | CDKN2A enhances chemokine expression. (A) Chemokine expression in CDKN2A Diploid and Deep deletion melanoma cell lines. (B) Cell cycle analysis of vehicle, double thymidine treated B16F0 cells and CDKN2A re-expressed B16F0 cells. (C) The mRNA expression of chemokine Ccl4, Ccl5, Cxcl9, Cxcl10, and Cxcl11 expression in control, CDKN2A re-expressed and double thymidine treated B16F0 cells. (D) ELISA analysis of the chemokine Ccl4, Ccl5, and Cxcl10 expression in control, CDKN2A re-expressed and double thymidine treated B16F0 cells. (E) The mRNA expression of chemokine Ccl4, Ccl5, Cxcl9, Cxcl10, and Cxcl10 in B16F0 cells arrested at G1/S and the cells arrested at G2/M phase. (F) The GSEA analysis of the gene expression in P38/MAPK and NF-kB pathway in patients with normal CDKN2A or CDKN2A deletion in TCGA-SKCM cohort. (G) The expression of protein in p38/MAPK and NF-kB pathway in B16F0 cells arrested at G1/S phase, S phase, G2/M phase with or without H2O2 treatment. (H) mRNA expression of chemokine Ccl4, Ccl5, Cxcl9, Cxcl10, and Cxcl11 in B16F0 cells treated with H2O2 or not. (I) The mRNA expression of genes downstream of p38/MAPK and NF-kB pathway in B16F0 cells arrested at G1/S phase, S phase, G2/M phase with or without H2O2 treatment. *p < 0.05, **p < 0.01, ***P < 0.001, ns, not significant.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBST and incubated with a primary antibody against Vinculin (1:1000, sc-73264, RRID : AB_1131292, Santa Cruz, USA), phospho-p65 (Ser536) (1:1000, #3031S, RRID : AB_330559, Cell Signaling Technology, Boston, USA), phospho-p38 MAPK (Thr180/Tyrl82) (1:1000, #4631S, RRID : AB_331765, Cell Signaling Technology), phospho-ATF2 (T71) (1:1000, BS4018, RRID : AB_1664091, Bioworld, China), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, #4370P, RRID : AB_2315112, Cell Signaling Technology), and IkBa (1:1000, #9242S, RRID : AB_331623, Cell Signaling Technology), CDK4 (1:500, sc-23896, RRID : AB_627239, Santa Cruz), E2F1 (1:1000, 12171-1-AP, RRID : AB_2096958, Proteintech), p16INK4 (1:500, sc-1661, RRID : AB_628067, Santa Cruz), Phospho-Histone H3 (1:1000, #9701, RRID : AB_331535, Cell Signaling Technology), Cyclin D1 (1:200, sc753, RRID : AB_2070433, Santa Cruz), Cyclin E1 (1:500, BS1086, RRID : AB_1663604, Bioworld), Cyclin B1 (1:1000, sc-245, RRID ;: AB_627338, Santa Cruz), at 4°C overnight.

Techniques: Expressing, Cell Cycle Assay, Control, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Biochemical and biophysical research communications

Article Title: AMPK activators contribute to maintain naïve pluripotency in mouse embryonic stem cells.

doi: 10.1016/j.bbrc.2018.11.164

Figure Lengend Snippet: Fig. 4. p38 is a functional downstream of AMPK. (A) p38 activation with AMPK activators. Western blot analysis shows protein expression of p38, phosphorylated p38 (Thr180/ Tyr182), a unique p38 downstream target mapkapk2 and phosphorylated mapkapk2 (Thr334). Note that p38 is activated commonly with AMPK activators. (B and C) FACS analysis for Rex1-GFPþcell appearance with a p38 inhibitor. p38i: SB203580 (10 mM). (D) Tetracycline-inducible (Tet-ON) constitutive active form of p38 (CA-p38) expression marked with mCherry. Western blot confirms dox-inducible activation of p38 pathway with CA-p38 expression. Doxycycline (1 mg/ml) treatment. (E) FACS analysis for Rex1-GFPþcell appearance after p38 activation.

Article Snippet: Please cite this article as: Y. Liu, J.K. Yamashita, AMPK activators contrib Biochemical and Biophysical Research Communications, https://doi.org/1 primary antibodies for following targets: phosphorylated-AMPK (Thr172, Cell Signaling (2531S) 1:1000), AMPK (Cell Signaling (2532S), 1:1000), phosphorylated-p38 (Thr180/Tyr182, Cell Signaling (9215S), 1:1000), p38 (Cell Signaling (9212S), 1:1000), phosphorylated-Mapkapk2 (Thr334, Cell Signaling (3007S), 1:1000), Mapkapk2 (Cell Signaling (3042S), 1:1000), b-actin (Sigma (A5441), 1:10000).

Techniques: Functional Assay, Activation Assay, Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hibiscus sabdariffa L.: A potent natural neuroprotective agent for the prevention of streptozotocin-induced Alzheimer's disease in mice.

doi: 10.1016/j.biopha.2020.110303

Figure Lengend Snippet: Fig. 6. Effects of Hibiscus extracts and celecoxib on the activity of p38MAPK (A), and COX-2 (B), and on concentration of mPGES-1 (C) and PGE2 (D). p38MAPK and COX-2 levels in celecoxib (30 mg/kg), white and red Hibiscus (200 mg/kg each) groups were decreased compared to STZ (3 mg/kg, ICV) group and increased compared to the normal group. mPGES-1 and PGE2levels were normalized in the three treated groups. Panel 4 represents immunoblot analysis shown in Fig. 6.A. Panel 5 represents immunoblot analysis shown in Fig. 6.B. Data are expressed as means ± S.D. The significance of the dif- ference between means was tested by ANOVA followed by Tukey Kramer multiple compar- isons test. * P < 0.05 vs normal; @ P < 0.05 vs STZ-treated group; # P < 0.05 vs celecoxib. n = 6 mice, DF = 29. For p38MAPK: F = 354.1, R2 = 0.9827. For COX-2: F = 350.5, R2 = 0.9825. For m-PGES-1: F = 430.4, R2 = 0.9857. For PGE2: F = 354.3, R2 = 0.9827.

Article Snippet: The antibodies used were rabbit BACE1 monoclonal [EPR19523] (abcam, ab183612), mouse Cox-2 (H-3) monoclonal (Santa cruze Inc, sc-376861), rabbit PSENEN polyclonal (Boster Biological Technology, A04504) and rabbit p38 MAPK polyclonal (Biorbyt Ltd., orb127559).

Techniques: Activity Assay, Concentration Assay, Western Blot