Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 2 Vitamin E downregulates EGFR and inactivates MAPK signaling pathway. A Venn diagram of the intersection of drug and disease targets. B Network diagram of gene-drug-disease interaction relationship. C KEGG enrichment analysis results of 30 candidate targets. D Box diagram of EGFR expression in COPD attained through microarray dataset GSE3320, wherein blue box on the left indicates normal tissue samples and red box on the right indicates COPD samples. E EGFR expression in lung tissues of rats after CS or vitamin E treatment was detected by immunohistochemistry. F EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 protein expression in lung tissues of rats after CS or vitamin E treatment was detected by Western blot. G Statistical diagram of Panel F. The data in the figure are measurement data, which are expressed by the mean ± standard deviation. The data of each group are analyzed by unpaired t-test, and the data among multiple groups are compared by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the control group, #p < 0.05 vs. the CS group, n = 6.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Expressing, Microarray, Immunohistochemistry, Western Blot, Standard Deviation, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 3 Vitamin E relieves CS-induced HBE cell damage. A RT-qPCR was used to assess the expression of EGFR in HBE cells of each group. B Western blot was used to assess the expression of EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 in HBE cells of each group. C Statistical map of Panel B. D CCK8 assay was used to detect the viability of HBE cells in each group. E Annexin V/PI staining was used to detect the apoptosis of HBE cells in each group. F ELISA was used to detect the expression of inflammatory factors in the supernatant of HBE cells of each group. G ROS level in HBE cells of each group. H The SOD activity and MDA content in HBE cells of each group. The data in the figure were all measurement data, expressed as the mean ± standard deviation. The data of each group were analyzed by unpaired t-test, and the comparison of data among multiple groups was performed by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the Vector group, #p < 0.05 vs. the vector + CS group, and $p < 0.05 vs. the vector + CS + vitamin E group.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Comparison, Plasmid Preparation

Journal: The FEBS journal

Article Title: Mitogen-activated protein kinase p38 and retinoblastoma protein signalling is required for DNA damage-mediated formation of senescence-associated heterochromatic foci in tumour cells.

doi: 10.1111/febs.12435

Figure Lengend Snippet: Fig. 6. p38 MAPK pathway was required in tumour cell SAHF formation. (A, D) Western blots of the indicated protein levels from MDA-MB-231 cells treated with 100 nM Dox or SN-38 for the indicated days. (B) Western blots of the indicated protein levels from young and senescent WI38 cells. (C) MDA-MB-231 cells were treated with or without p38 MAPK pathway inhibitor SB203580 (10 lM) for 2 h, then 100 nM Dox was added for 1 + 7 days. The percentage of cells with DAPI foci was determined.

Article Snippet: The antibodies used to probe the blots were: rabbit anti-H3K9me3 (Millipore), mouse anti-HP1c (Millipore), rabbit anti-Rb (BD Pharmingen), rabbit anti-phospho-Rb (pSer807/811) (Sigma), rabbit anti-p21 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-p38 (CST, Danvers, MA, USA), rabbit anti-phospho-p38 (Thr180/Tyr182) (CST), rabbit anti-p44/ p42 (CST), rabbit anti-phospho-p44/p42 (CST), rabbit anti-SAPK/JNK (CST), rabbit anti-phospho-SAPK/JNK (CST), rabbit anti-HBP1 (Santa Cruz) and mouse anti-bactin (Sigma).

Techniques: Western Blot

Journal: The FEBS journal

Article Title: Mitogen-activated protein kinase p38 and retinoblastoma protein signalling is required for DNA damage-mediated formation of senescence-associated heterochromatic foci in tumour cells.

doi: 10.1111/febs.12435

Figure Lengend Snippet: Fig. 7. HBP1 was involved in SAHF formation. (A) The mRNA and protein levels of HBP1 were detected by real-time RT-PCR and western blotting. (B) MDA-MB-231 cells were treated with or without p38 MAPK pathway inhibitor SB203580 for 2 h, and then 100 nM Dox was added for 48 h. The mRNA level of HBP1 was detected by real-time RT-PCR. (C) The expression of the indicated protein was determined by western blotting in A549 cells after 200 nM Dox treatment for the indicated number of days. (D) The indicated protein levels from MDA- MB-231, A549 and WI38 cells after infection with control (Con) or H-RasV12 retrovirus for 8 days. (E) MDA-MB-231 cells were infected with control (Con) or HBP1 short hairpin RNA plasmid, then treated with 100 nM Dox for 1 + 7 days, and the percentage of cells with DAPI foci was counted. (F) Rb and HBP1 existed in the same complex. MDA-MB-231 cells were treated with 100 nM Dox for 1 + 3 days, whole-cell extracts were prepared and immunoprecipitated with anti-IgG, anti-HBP1 or anti-Rb antibodies, and incubated with protein A agarose beads. Immunoprecipitates were subjected to immunoblotting analysis with anti-Rb and anti-HBP1 sera. Western blots of the whole-cell extracts were detected with anti-Rb and anti-HBP1 sera (Input). (G) HBP1 regulated the incorporation of Rb on the target genes of E2F. MDA-MB- 231 cells were transfected with HBP1 siRNA vector for 48 h, then treated with or without 100 nM Dox for another 24 h and harvested for ChIP assays. Samples were immunoprecipitated with anti-Rb serum, and the precipitated DNA fragments were amplified using real-time PCR. Each pair of primer was used to amplify the DNA fragments in the immunoprecipitated samples or the non-immunoprecipitated (input) samples; the ratio was further normalized to the levels in cells transduced with empty vector and without drug treatment.

Article Snippet: The antibodies used to probe the blots were: rabbit anti-H3K9me3 (Millipore), mouse anti-HP1c (Millipore), rabbit anti-Rb (BD Pharmingen), rabbit anti-phospho-Rb (pSer807/811) (Sigma), rabbit anti-p21 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-p38 (CST, Danvers, MA, USA), rabbit anti-phospho-p38 (Thr180/Tyr182) (CST), rabbit anti-p44/ p42 (CST), rabbit anti-phospho-p44/p42 (CST), rabbit anti-SAPK/JNK (CST), rabbit anti-phospho-SAPK/JNK (CST), rabbit anti-HBP1 (Santa Cruz) and mouse anti-bactin (Sigma).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Infection, Control, shRNA, Plasmid Preparation, Immunoprecipitation, Incubation, Transfection, Real-time Polymerase Chain Reaction, Transduction

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: DLEU2 activates the MAPK-signaling pathway via enhancing RARB promoter methylation. (a-b) target genes of RARB showing an over 0.5 correlation coefficient with RARB and the KEGG pathway analysis based on these genes; (c) RARB expression in cells after oe-DLEU2 or sh-DLEU2 transfections detected by RT-qPCR (two-way ANOVA) (DLEU2: SW480: oe-DLEU2 vs. NC: p < 0.0001; sh-DLEU2 vs. NC: p = 0.0025; HT29: oe-DLEU2 vs. NC: p < 0.0001; sh-DLEU2 vs. NC. p = 0.0087; RARB: SW480: oe-DLEU2 vs. NC: p = 0.0024, sh-DLEU2 vs. NC: p < 0.0001; HT29: oe-DLEU2 vs. NC: p = 0.001, sh-DLEU2 vs. NC: p < 0.0001); (d) methylation level in the RARB promoter examined by qMSP (two-way ANOVA) (SW480: oe-DLEU2 vs. NC: p < 0.0001, sh-DLEU2 vs. NC p = 0.0013; HT29: oe-DLEU2 vs. NC p < 0.0001, sh-DLEU2 vs. NC: p = 0.0033); (e) phosphorylation and protein levels of Raf, p38, and ERK in CRC cells determined by immunoblot analysis (two-way ANOVA) (see p values in ). Repetition = 3.

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Methylation, Expressing, Transfection, Quantitative RT-PCR, Phospho-proteomics, Western Blot

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: P values of the phosphorylation extent of the Raf, p38, and ERK between the oe-DLEU2 and NC groups in SW480 and HT29 cells

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Phospho-proteomics

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: RARB silencing blocks the inhibiting role of sh-DLEU2 in CRC cells. (a) RARB expression in SW480 and HT29 cells after sh-RARB transfection detected by RT-qPCR (DLEU2: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.9657; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.8714; RARB: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0006; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0001); (b) viability of SW480 and HT29 cells examined by the CCK-8 method (two-way ANOVA) (SW480: 0 h: p = 0.9931; 24 h: p = 0.4837; 48 h: p = 0.0069; 72 h: p = 0.0001; HT29: 0 h: p = 0.9947; 24 h: p = 0.2835; 48 h: p = 0.0013; 72 h: p < 0.0001); (c-d) migration and invasiveness of SW480 and HT29 cells measured by Transwell assays, respectively (two-way ANOVA) (C: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0005; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB p = 0.0002; D: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.001; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0006); (e) apoptosis of SW480 and HT29 cells detected by flow cytometry (two-way ANOVA) (SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0001; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0003); (f) phosphorylation and protein levels of Raf, p38, and ERK in CRC cells determined by immunoblot analysis (two-way ANOVA) (see p values in ); (g) p38 phosphorylation in CRC cells after Dehydrocorydaline treatment examined by immunoblot analysis (sh-DLEU2 + DMSO vs. sh-DLEU2 + Dehydrocorydaline: SW480: p = 0.0006; HT29: p = 0.0052); (h) proliferation of cells examined by the CCK-8 assay (sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: 0 h: p = 0.9990, 24 h: p = 0.6695, 48 h: p < 0.0001, 72 h: p < 0.0001; HT29: 0 h, p = 0.9997, 24 h: p = 0.0603, 48 h: p < 0.0001, 72 h, p < 0.0001); (i-j) migration (i, sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: p < 0.0001; HT29: p < 0.0001) and invasiveness (j, sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: p = 0.001; HT29: p = 0.0017) of cells analyzed by Transwell assays; (k) apoptosis of cells examined by flow cytometry (sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: p = 0.0005; HT29: p = 0.0004). Repetition = 3.

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Migration, Flow Cytometry, Phospho-proteomics, Western Blot

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: P values of the phosphorylation extent of the Raf, p38, and ERK between the sh-DLEU2 + sh-NC and sh-DLEU2 + sh-RARB groups in SW480 and HT29 cells

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

doi: 10.1074/jbc.M513397200

Figure Lengend Snippet: FIGURE 1. A, Erk8 expression in human thyroid cancer cell lines. After RNA extraction, quantitative RT-PCR was performed to evaluate Erk8 expression in human ARO, Kat4, and Cal62 cell lines. As a control (contr) for the specificity of the primers, rat Pc cells were also analyzed. B, schematic representation of the wild-type RET protein and of its activated form, the RET/PTC3 oncogene. The position of three tyrosine autophosphorylation sites and of important protein domains are also shown. Numbers indicating RET/PTC3 tyrosine residues correspondtotheirpositioninthewild-typeRETreceptor.SP,signalpeptide;TMD,trans-membranedomain;TK,tyrosinekinase;Y,tyrosine;RFG,RETfusedgene.C,analysisofHA-Erk8 activation in 293T cells cotransfected with the RET/PTC3 (PTC3) and Src YF expression vectors and analyzed by Western blot with anti-phospho MAPK (-P-MAPK) antisera. Activation of endogenous Erk2 by RET/PTC3 and Src YF was used as an additional control for the activity of the two oncogenes. The expression of HA-Erk8 (-HA panel), Erk2 (-Erk2 panel), RET/PTC3 (-RET panel), and Src YF (-Src panel) was also confirmed. P, phospho. D, analysis of HA-Erk8 activation in 293T cells cotransfected with the increasing amounts of RET/MEN2B(MEN2B)andRET/PTC3(PTC3)expressionvectorsandanalyzedbyWesternblotwithanti-phosphoMAPKantisera.TheexpressionofHA-Erk8(-HApanel)wasconfirmed. Control, cells transfected with -galactosidase; , anti.

Article Snippet: Antibodies—As primary antibodies, we used rabbit polyclonal antisera to Erk2 (C-14) (Santa Cruz Biotechnology), phospho-MAPK (p42/ p44) (Cell Signaling), RET and phospho-RET (phospho-Tyr905) (11), and mouse monoclonal antibodies to enhanced green fluorescent protein, AU5, and HA epitopes (Berkeley Antibody Company), Src (Ab-1; Oncogene Science), c-Abl (Pharmingen), and phospho-tyrosine, PY (Santa Cruz Biotechnology and Upstate Biotechnology).

Techniques: Expressing, RNA Extraction, Quantitative RT-PCR, Control, Activation Assay, Western Blot, Activity Assay, Transfection

Journal: The Journal of biological chemistry

Article Title: Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

doi: 10.1074/jbc.M513397200

Figure Lengend Snippet: FIGURE 3. Stimulation of Erk8 activation by RET/PTC3 (PTC3) and by tyrosine-mu- tatedformsoftheoncogene,in293Tcellsco-transfectedwithanexpressionvector for HA-Erk8 (-P-MAPK panel). Activation of endogenous Erk2 by RET/PTC3 and its different mutated forms was used as a parallel control for the activity of the oncogenes (-P-MAPK panel). The expression of HA-Erk8 (-HA panel), Erk2 (-Erk2 panel), and RET/ PTC3 wild-type and mutants (-RET panel) was also confirmed. , anti; P, phospho; con- trol, cells transfected with -galactosidase; Kin dead, kinase dead.

Article Snippet: Antibodies—As primary antibodies, we used rabbit polyclonal antisera to Erk2 (C-14) (Santa Cruz Biotechnology), phospho-MAPK (p42/ p44) (Cell Signaling), RET and phospho-RET (phospho-Tyr905) (11), and mouse monoclonal antibodies to enhanced green fluorescent protein, AU5, and HA epitopes (Berkeley Antibody Company), Src (Ab-1; Oncogene Science), c-Abl (Pharmingen), and phospho-tyrosine, PY (Santa Cruz Biotechnology and Upstate Biotechnology).

Techniques: Activation Assay, Control, Activity Assay, Expressing, Transfection

Journal: The Journal of biological chemistry

Article Title: Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

doi: 10.1074/jbc.M513397200

Figure Lengend Snippet: FIGURE 5. A, analysis of HA-Erk8 activation in 293T cells cotransfected with Src YF, an activated form of c-Abl, the Bcr/Abl oncogenic fusion protein. Samples were analyzed by Western blot with anti-phospho MAPK (-P-MAPK) antisera. The expression of HA-Erk8 (-HA panel) and Bcr/Abl (-Abl panel) was confirmed. Control, cells transfected with -galactosidase;,anti.B,analysisofErk8activation(-P-MAPKpanel)in293Tcellstrans- fected with HA-Erk8, RET/PTC3, and increasing concentrations (100, 250, and 500 ng) of the dominant negative Abl-KD. The expression of HA-Erk8 (-HA panel) and of Abl-KD (-AU5 panel) was also confirmed. C, stimulation of c-Abl phosphorylation by RET/PTC3 (PTC3) and by tyrosine-mutated forms of the oncogene. 293T cells were co-transfected with an expression vector for AU5-Abl-KD together with plasmids for RET/PTC3 and its tyrosine mutants. Samples were next immunoprecipitated (IP) by -AU5 antibodies and then analyzed by -phospho-tyrosine antibodies (-PY). The expression of AU5-Abl-KD (-AU5 panel) and RET/PTC3 wild-type and mutants (-RET panel) was also confirmed. WB, Western blot.

Article Snippet: Antibodies—As primary antibodies, we used rabbit polyclonal antisera to Erk2 (C-14) (Santa Cruz Biotechnology), phospho-MAPK (p42/ p44) (Cell Signaling), RET and phospho-RET (phospho-Tyr905) (11), and mouse monoclonal antibodies to enhanced green fluorescent protein, AU5, and HA epitopes (Berkeley Antibody Company), Src (Ab-1; Oncogene Science), c-Abl (Pharmingen), and phospho-tyrosine, PY (Santa Cruz Biotechnology and Upstate Biotechnology).

Techniques: Activation Assay, Western Blot, Expressing, Control, Transfection, Dominant Negative Mutation, Phospho-proteomics, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

doi: 10.1074/jbc.M513397200

Figure Lengend Snippet: FIGURE 7. A, Erk8 and Erk8 expression in transiently transfected 293T cells. Control, cells transfected with -galactosidase; , anti. B, comparison of Erk8 and Erk8 nucleotide and protein sequences in the region of the alternative splicing. C, schematic representation of Erk8 and Erk8 protein structures. The relative position of important residues and protein domains is indicated. D, analysis of Erk8 and Erk8 activation in 293T cells cotransfected with the RET/PTC3 (PTC3) and Src YF expression vectors. Activation of endogenous Erk2 by RET/PTC3 and Src YF was used as an additional control for the activity of the two oncogenes. The expression of HA-Erk8, HA-Erk8 (-HA panel), and Erk2 (-Erk2 panel) was also confirmed. E, analysis of HA-Erk8 activation in 293T cells cotransfected with the RET/PTC3 (PTC3) and Src YF expression vectors and analyzed by Western blot with anti-phospho MAPK (-P-MAPK) antisera. To be able to appreciate Erk8 low levels of phosphorylation, polyvinylidene difluoride membrane was exposed 10 min after ECL reaction. The expression of HA-Erk8 (-HA panel) was confirmed. F, analysis of Erk8 activation in 293T cells cotransfected with Bcr/Abl and Src YF expression vectors. The expression of HA-Erk8 (-HA panel) was confirmed.

Article Snippet: Antibodies—As primary antibodies, we used rabbit polyclonal antisera to Erk2 (C-14) (Santa Cruz Biotechnology), phospho-MAPK (p42/ p44) (Cell Signaling), RET and phospho-RET (phospho-Tyr905) (11), and mouse monoclonal antibodies to enhanced green fluorescent protein, AU5, and HA epitopes (Berkeley Antibody Company), Src (Ab-1; Oncogene Science), c-Abl (Pharmingen), and phospho-tyrosine, PY (Santa Cruz Biotechnology and Upstate Biotechnology).

Techniques: Expressing, Transfection, Control, Comparison, Alternative Splicing, Activation Assay, Activity Assay, Western Blot, Phospho-proteomics, Membrane

Journal: The Journal of biological chemistry

Article Title: Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

doi: 10.1074/jbc.M513397200

Figure Lengend Snippet: FIGURE 8. A, schematic representation of Erk8 protein and of its derivative C-terminal deletion mutants. The relative position of the TEY and PXXP motifs is also shown. B, analysis of theactivationofErk8andofitsderivativeC-terminaldeletionmutantsin293TcellscotransfectedwithRET/PTC3(PTC3)andBcr/Ablexpressionvectors.TheexpressionofHA-Erk8and of the different deletion mutants (-HA panel) was confirmed. C, analysis of HA-Erk8 activation in 293T cells cotransfected with Src YF and activated forms of c-Abl, Bcr/Abl, and Abl Act. Samples were analyzed by Western blot with anti-phospho MAPK (-P-MAPK) antisera. The expression of HA-Erk8 (-HA panel) was confirmed. Control, cells transfected with -galactosidase; , anti.

Article Snippet: Antibodies—As primary antibodies, we used rabbit polyclonal antisera to Erk2 (C-14) (Santa Cruz Biotechnology), phospho-MAPK (p42/ p44) (Cell Signaling), RET and phospho-RET (phospho-Tyr905) (11), and mouse monoclonal antibodies to enhanced green fluorescent protein, AU5, and HA epitopes (Berkeley Antibody Company), Src (Ab-1; Oncogene Science), c-Abl (Pharmingen), and phospho-tyrosine, PY (Santa Cruz Biotechnology and Upstate Biotechnology).

Techniques: Activation Assay, Western Blot, Expressing, Control, Transfection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A : cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. *** P < 0.001. B : this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C : PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. ** P < 0.005. D : PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. * P < 0.05; ** P < 0.005. E : PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. *** P < 0.005. F : PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. *** P < 0.001.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Isolation, Derivative Assay, Inhibition, Thymidine Incorporation Assay, Western Blot, Activation Assay, Proliferation Assay, Positive Control, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Increased expression of p38 MAPK α-isoform in animal models of PH. A : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the BCA method. Equal concentrations were then loaded on a gel and blotted for p38 MAPKα and β-actin for loading control. There are 3 wells for each condition. Immunoblot shown is best representative of 3 experiments using 3 different animals with each condition. B : densitometry of immunoblot in A . Values are mean arbitrary values from 3 immunoblots expressed relative to the value for β-actin. * P < 0.05 by ANOVA. C : lung sections (5 mm) were prepared from normal, CH, and MCT animals. Sections were stained for p38 MAPKα using 1:400 dilution. Magnification: ×20. Bar represents = 150 mm. Arrows identify blood vessels. D : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then used in a p38 MAPK activity assay using immunoprecipitation and the phosphorylation of activating transcription factor-2 (ATF-2) as a read out. Immunoblot shown is representative of 3 experiments using 3 different animals. E and F : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phosphorylated p38 MAPK and β-actin for loading control. Immunoblot shown is representative of 3 experiments using 3 different animals with each condition. Densitometry is shown for remaining blots. * P < 0.05.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Expressing, Protein Concentration, Control, Western Blot, Staining, Activity Assay, Immunoprecipitation, Phospho-proteomics

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH is prevented by administration of SB203580, a p38 MAPKα inhibitor. A and B : animals were exposed to a hypobaric hypoxic environment for 2 wk. Some animals received daily injections of SB203580. Hemodynamics ( A ) and hematocrit ( B ) were measured after 2 wk. RVSP, right ventricular (RV) systolic pressure. Data represent mean values ± SE. Total animals n = 5–6 per group. C : hearts were isolated from the animals and the RV was dissected out from the left ventricle (LV) and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. The total RV weight was also plotted. Values are means ± SD; n = 5 per group. ** P < 0.05. D : noninvasive systemic blood pressure taken by tail-cuff measurement. E and F : rats were exposed to hypoxia ± SB203580. The lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five random fields per slide were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized and are presented as numbers per total number of vessels present in each field. Groups analyzed by ANOVA for overall change with posttest analysis; n = 5 animals. *** P < 0.001, for A – C and E .

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Isolation, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH in 2 in vivo animal models is reversed by the administration of SB203580, a p38 MAPKα inhibitor. A : animals were exposed to a hypobaric hypoxic environment for 2 wk and then p38 MAPK inhibition was commenced. Hemodynamics and RVSP were measured after 4 wk. Data represent mean values ± SE. Total animals n = 5–6 per group. ** P < 0.01; *** P < 0.001, for normal relative to all other conditions. B : hearts were isolated form the animals and the RV dissected out from the LV and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. Values are means ± SE; n = 6–7. ** P < 0.01; *** P < 0.005. C : hematocrit ratio. D : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. ** P < 0.01; *** P < 0.001. E : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 7 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. ** P < 0.01; *** P < 0.001. F and G : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 6–7 per group. * P < 0.05; *** P < 0.001, for F . * P < 0.05; ** P < 0.01, for G . H : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. * P < 0.05; *** P < 0.001. I : lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 MCT control.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: In Vivo, Inhibition, Isolation, Staining, Control, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH in a reversal strategy in 2 in vivo animal models by administration of PH-797804, a more selective p38 MAPKα inhibitor. A and B : animals were exposed to CH and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RV hypertrophy (RVH) were measured after 4 wk. Data represent mean values ± SE. * P < 0.05; ** P < 0.01 for A . ** P < 0.01; *** P < 0.001, for B . C : the lungs were removed after experiment and sections (5 mm) were cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05 for complete muscularized group in drug-treated vs. day 14 hypoxic control. D : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. E and F : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 14–15 per group. *** P < 0.001 for E . * P < 0.05; ** P < 0.01 for F . G : the lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05, for complete muscularized group in drug-treated vs. day 14 hypoxic control. H : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for drug-treated vs. day 14 MCT control.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: In Vivo, Inhibition, Staining, Control, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B : supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. * P < 0.05 ** P < 0.005. C : PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D : PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. ** P < 0.01; *** P < 0.01, for C and D . E : PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. * P < 0.05; ** P < 0.005; *** P < 0.0001. F : PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. **** P < 0.0001. G : PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. ** P < 0.01. H and I : PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. *** P < 0.005; **** P < 0.001, for I .

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, DNA Synthesis, Marker

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A : lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B : lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05 ** P < 0.01. C and D : lungs from CH 28-day controls and SB203580 -treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. ** P < 0.001. E : lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05; ns is not significant by ANOVA. F : PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. *** P < 0.005. G : PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. * P < 0.01; *** P < 0.005. H : fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. ** P < 0.01, *** P < 0.001.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Inhibition, In Vivo, Isolation, Protein Concentration, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Cell Culture, Derivative Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Phospho-p38 MAPK and p38 MAPKα expression in explanted lungs from patients with idiopathic pulmonary arterial hypertension (IPAH). A : sections of 5 mm were taken. Then, normal control lung and IPAH lung are stained for phospho-p38 MAPK at dilution of 1:300. The isotype on IPAH lung is also shown. This dilution was optimally assessed for. Objective: ×20. Bar = 150 mm. B : high-power microscopy shows that there is strong staining for phospho-p38 MAPK in the intima, media, and the adventitia (arrows). Objective lens: ×20 and ×40. Bar = 50 mm. C : sections of control lung ( A ) and IPAH lung ( B ) were stained for p38 MAPKα at dilution of 1:300. Objective lens × 20. Bar = 150 mm. D : staining for p38 MAPKa showed increased cytosolic staining in the IPAH lung ( right ) compared with control lung ( left ). E : high-power view (×40) of staining with isotype and p38 MAPKα in a vessel in IPAH lung. This shows staining throughout the vessel layers but especially in adventitia and fibroblast cells (arrow). Bar = 50 mm. F : low-power view of a plexiform lesion. Staining for p38 MAPKα using 1:300 dilution. Objective lens × 20. Bar = 150 mm. G : histological scoring shows increased p38 MAPKα staining throughout the vascular wall. With the use of a well-validated histological scoring system (Allred), the vascular wall cells were scored for intensity of staining. The intensity multiplied by the number of vessels with that intensity determines the values. Values shown are from 5 random high-power fields from 2 slides. **** P < 0.0001 by ANOVA. ** P < 0.001 for individual IPAH vs. control columns.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Expressing, Control, Staining, Microscopy

Journal: Cell Death & Disease

Article Title: Bortezomib-inducible long non-coding RNA myocardial infarction associated transcript is an oncogene in multiple myeloma that suppresses miR-29b

doi: 10.1038/s41419-019-1551-z

Figure Lengend Snippet: a–e U266 cells were pretreated with the selective pharmacological inhibitors U0126 (ERK, 50 μM), SP600125 (JNK, 50 μM), Bay-11-7082 (NF-κB, 10 μM), SB203580 (p38, 50 μM), or PF-04965842 (Jak1, 50 nM), and were then treated with BTZ at 40 nM for 12 h. MIAT expression levels were determined using qRT-PCR. f Western blotting analyses of Stat1 and phosphorylated Stat1 after overexpression or knockdown in U266 cells. g Effects of Stat1 overexpression on MIAT expression in U266 cells. h Effects of Stat1 knockdown on MIAT expression in U266 cells. i Effects of p38 overexpression on MIAT expression in U266 cells pretreated with sh-NC or sh-Stat1. j Luciferase reporter constructs containing the MIAT promoter were co-transfected into U266 cells with the internal control plasmid pRL-TK, and with sh-NC or sh-Stat1, and were then subjected to BTZ challenge (40 nM, 12 h). Relative luciferase activities are expressed as percentages of those in the control group. k Cell lysates from U266 cells were used for RIP with antibodies against stat1, stat3, or NF-κB. MIAT expression levels were detected using qRT-PCR. IgG was used as a negative control. l BTZ induced Stat1 phosphorylation; data are presented as means ± standard errors of the mean from three independent experiments; * P < 0.05; two-tailed pairwise comparisons were made with Student’s t -test

Article Snippet: Following separation on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, proteins were transferred onto polyvinylidene (PVDF) membranes and were immunoblotted with the following primary antibodies: phospho-Stat1 (Tyr701) antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 9167, Cell Signaling Technology), phospho-Stat1 (Ser727) antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 8826, Cell Signaling Technology), Stat1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 14994, Cell Signaling Technology), p38 MAPK antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 8690, Cell Signaling Technology), Bcl-2 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 4223, Cell Signaling Technology), Bak1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 12105, Cell Signaling Technology), cleaved PARP antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 5625, Cell Signaling Technology), total PARP antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 9532, Cell Signaling Technology), Mcl-1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 94296, Cell Signaling Technology), CDK-6 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 13331, Cell Signaling Technology), PSME4 antibody (Rabbit polyclonal, diluted at 1:1000, cat no. ABIN5533127, Abgent Biotech. (SuZhou) Co. Ltd., Suzhou, China), SP-1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 9389, Cell Signaling Technology), and GAPDH antibody (Rabbit polyclonal, diluted at 1:1000, cat no. 5174, Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, Luciferase, Construct, Transfection, Control, Plasmid Preparation, Negative Control, Phospho-proteomics, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Insulin-like Growth Factor-binding Protein-3 Potentiates Epidermal Growth Factor Action in MCF-10A Mammary Epithelial Cells

doi: 10.1074/jbc.m210739200

Figure Lengend Snippet: FIG. 6. IGFBP-3 potentiates EGF-stimulated p44/42 and p38 MAPK phosphorylation. Panel A, confluent MCF-10A cells in six- place wells were incubated in the absence (lanes 1 and 2) or presence (lane 3) of 10 ng/ml IGFBP-3 for 24 h. Media were removed and replaced by serum-free medium (lane 1) or medium containing 1 ng/ml EGF (lanes 2 and 3) for 8 min. Cell lysates were prepared as described under “Experimental Procedures” and then subjected to 12% SDS-PAGE and Western blotting for the indicated proteins using phosphospecific anti- bodies, followed by ECL detection. Panel B shows densitometric quan- titation of the blots, where the EGF and EGF IGFBP-3 bands have been expressed as a percentage increase above control (no EGF or IGFBP) for each protein.

Article Snippet: In Vitro Kinase Assays—Activity of p38 MAPK and p44/42 MAPK in lysates from IGFBP-3- and EGF-treated MCF-10A cells was determined by assessing phosphorylation of Elk-1 (for p44/42 MAPK) and Atf-2 (for p38 MAPK) using reagents from Cell Signaling (Beverley, MA).

Techniques: Phospho-proteomics, Incubation, SDS Page, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Insulin-like Growth Factor-binding Protein-3 Potentiates Epidermal Growth Factor Action in MCF-10A Mammary Epithelial Cells

doi: 10.1074/jbc.m210739200

Figure Lengend Snippet: FIG. 7. IGFBP-3 potentiates EGF stimulation of p44/42 and p28 MAP kinase activities. Panel A, confluent MCF-10A cells in six-place wells were incubated in the absence (lanes 1 and 2) or presence of 10 ng/ml (lane 3) or 100 ng/ml IGFBP-3 (lane 4) for 24 h. Media were removed and replaced by serum-free medium (lane 1) or medium con- taining 1 ng/ml EGF (lanes 2–4) for 8 min. Cell lysates were assayed for p44/42 MAP kinase activity (using Elk-1 as substrate) and p38 MAP kinase activity (using Atf-2 as substrate) as described under “Experi- mental Procedures.” Panel B shows densitometric quantitation of data derived from two experiments (mean S.D.), where the EGF (lane 2) and EGFIGFBP-3 (lanes 3 and 4) bands have been expressed as a percentage increase above control (no EGF or IGFBP; lane 1) for each protein. *, p 0.05 compared with EGF alone.

Article Snippet: In Vitro Kinase Assays—Activity of p38 MAPK and p44/42 MAPK in lysates from IGFBP-3- and EGF-treated MCF-10A cells was determined by assessing phosphorylation of Elk-1 (for p44/42 MAPK) and Atf-2 (for p38 MAPK) using reagents from Cell Signaling (Beverley, MA).

Techniques: Incubation, Activity Assay, Quantitation Assay, Derivative Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Insulin-like Growth Factor-binding Protein-3 Potentiates Epidermal Growth Factor Action in MCF-10A Mammary Epithelial Cells

doi: 10.1074/jbc.m210739200

Figure Lengend Snippet: FIG. 8. Potentiation of EGF signaling by IGFBP-3 is blocked by inhibitors of p44/42 and p38 MAP kinases. MCF-10A cells were incubated with EGF (0.1 ng/ml) and the indicated dose of IGFBP-3 in the presence of 20 M PD98059 (panel A), 10 M SB203580 (panel B), or 10 M LY294002 (panel C). DNA synthesis was determined after 24 h, as described under “Experimental Procedures.” Results are shown rel- ative to thymidine incorporation in the presence of EGF inhibitor, and are mean S.E. of pooled data from three experiments, each carried out in quadruplicate. *, p 0.05; **, p 0.01 compared with EGF inhibitor in the absence of IGFBP-3.

Article Snippet: In Vitro Kinase Assays—Activity of p38 MAPK and p44/42 MAPK in lysates from IGFBP-3- and EGF-treated MCF-10A cells was determined by assessing phosphorylation of Elk-1 (for p44/42 MAPK) and Atf-2 (for p38 MAPK) using reagents from Cell Signaling (Beverley, MA).

Techniques: Incubation, DNA Synthesis

Journal: PLoS ONE

Article Title: VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca 2+ /Calcineurin and Protein Kinase C-δ

doi: 10.1371/journal.pone.0011435

Figure Lengend Snippet: ( A ) Exon structure of RCAN1, showing the alternative first exons used by the RCAN1.1 and RCAN1.4 isoforms. ( B ) HDMEC were stimulated with VEGF-A (50 ng/ml) for 1 hour, and the increase in expression of RCAN1.1 and RCAN1.4 mRNA, relative to the level of each mRNA in unstimulated (basal) cells, determined by RT-qPCR. Results were normalised to β-actin mRNA expression. Data shows mean ± SD (n = 3). **P<0.01, versus Basal. ( C ) HDMEC were stimulated with VEGF-A (50 ng/ml), VEGF-B (50 ng/ml), VEGF-E (50 ng/ml) or VEGF-B and –E (50 ng/ml each) for 1 hour and the expression of RCAN1.4 mRNA determined by RT-qPCR. Results were normalised to β-actin mRNA expression. Data shows mean ± SD (n = 3). **P<0.01, versus Basal. ( D ) HDMEC were stimulated with agonists as in 1 C for 10 mins or 3 hours and the expression levels of RCAN1.4 (3 hour stimulation), and phospho-VEGFR2 (pVEGFR2) proteins determined by SDS-PAGE followed by western blotting. Actin was used as a loading control. Numbers below the blots denote the relative density of each band. Results are representative of 3 experiments. ( E ) HDMEC were stimulated with VEGF-A (50 ng/ml), FGF-2 (50 ng/ml), EGF (50 ng/ml), HGF (50 ng/ml), LPA (10 µM), A23187 (5 µM) and PMA (100 nM) for 1 hour, and the expression of RCAN1.4 mRNA, relative to unstimulated (basal) cells, determined by RT-qPCR. Results were normalised to β-actin mRNA expression. Data shows mean ± SD (n = 3). **P<0.01, versus Basal. ( F ) HDMEC were stimulated with agonists as in 1 C for 10 mins or 3 hours and the expression levels of RCAN1.4 (3 hour stimulation), phospho–PLCγ (p-PLCγ), NFAT and phospho-p44/42 MAPK (p-MAPK) proteins determined by SDS-PAGE followed by western blotting. Actin was used as a loading control. Numbers below the blots denote the relative density of each band. Results are representative of 3 experiments.

Article Snippet: Proteins were detected using the following antibodies; RCAN1 (D6694; Sigma), NFAT1 (610702; BD Transductions Labs), phospho-p44/42 MAPK (Thr202/Tyr204; #4377), phospho-PKC (pan; #9371), phospho-PLCγ (Tyr 783; #2821), PKCα (#2056), PKCδ (∼2058; all Cell Signalling Technology), PKCε (06-991; Upstate), PKCη (sc-215), colony stimulating factor-1 receptor (CSF-1R; sc-13949) and Actin (sc-1625; all Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, SDS Page, Western Blot, Control

Journal: PLoS ONE

Article Title: VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca 2+ /Calcineurin and Protein Kinase C-δ

doi: 10.1371/journal.pone.0011435

Figure Lengend Snippet: ( A ) Structure of chimeric receptors used to transfect HDMEC. ( B ) HDMEC were transfected with chimeric receptors and stimulated with CSF-1 (C; 50 ng/ml) or VEGF-A (V; 50 ng/ml) for 10 mins. The levels of CSF-1R, phospho-PLCγ (p-PLCγ) and phospho-p44/42 MAPK (pMAPK) were determined by SDS-PAGE followed by western blotting. Actin was used as a loading control. Numbers below the blots denote the relative density of each band. Results are representative of 3 experiments. ( C ) HDMEC were transfected with chimeric receptors, stimulated with CSF-1 (50 ng/ml) or VEGF-A (50 ng/ml) for 1 hour, and the expression of RCAN1.4 mRNA, relative to unstimulated vector transfected cells, determined by RT-qPCR. Results were normalised to β-actin mRNA expression. Data shows mean ± SD (n = 3). **P<0.01, versus unstimulated vector control.

Article Snippet: Proteins were detected using the following antibodies; RCAN1 (D6694; Sigma), NFAT1 (610702; BD Transductions Labs), phospho-p44/42 MAPK (Thr202/Tyr204; #4377), phospho-PKC (pan; #9371), phospho-PLCγ (Tyr 783; #2821), PKCα (#2056), PKCδ (∼2058; all Cell Signalling Technology), PKCε (06-991; Upstate), PKCη (sc-215), colony stimulating factor-1 receptor (CSF-1R; sc-13949) and Actin (sc-1625; all Santa Cruz Biotechnology).

Techniques: Transfection, SDS Page, Western Blot, Control, Expressing, Plasmid Preparation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca 2+ /Calcineurin and Protein Kinase C-δ

doi: 10.1371/journal.pone.0011435

Figure Lengend Snippet: HDMEC were stimulated with VEGF-A (50 ng/ml), FGF-2 (50 ng/ml) PMA (100 nM) or A23187 (5 µM) for the time points indicated. The expression levels of phospho-PLCγ (p-PLCγ), phospho-PKC (p-PKC) and phospho-p44/42 MAPK (p-MAPK) were determined by SDS-PAGE followed by western blotting. Actin was used as a loading control. Graphs show the relative expression of each phosphorylated PKC isoform in response to either VEGF-A, FGF-2 or PMA, as determined from the relative density of the bands compared to the unstimulated (basal) condition. Results are representative of 3 experiments.

Article Snippet: Proteins were detected using the following antibodies; RCAN1 (D6694; Sigma), NFAT1 (610702; BD Transductions Labs), phospho-p44/42 MAPK (Thr202/Tyr204; #4377), phospho-PKC (pan; #9371), phospho-PLCγ (Tyr 783; #2821), PKCα (#2056), PKCδ (∼2058; all Cell Signalling Technology), PKCε (06-991; Upstate), PKCη (sc-215), colony stimulating factor-1 receptor (CSF-1R; sc-13949) and Actin (sc-1625; all Santa Cruz Biotechnology).

Techniques: Expressing, SDS Page, Western Blot, Control

Journal: PLoS ONE

Article Title: VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca 2+ /Calcineurin and Protein Kinase C-δ

doi: 10.1371/journal.pone.0011435

Figure Lengend Snippet: ( A ) HDMEC were transfected with a mixture of two siRNAs to RCAN1 or non-silencing (N.S.) siRNA and the knockdown of RCAN1.4 and effects on phospho-PLCγ (p-PLCγ) and phospho-p44/42 MAPK (pMAPK) were determined by SDS-PAGE followed by western blotting. Actin was used as a loading control. Numbers below the blots denote the relative density of each band. Results are representative of 3 experiments. ( B ) HDMEC were transfected with a mixture of two RCAN1 siRNAs and stimulated with VEGF-A (50 ng/ml) for 1 hour. Expression of COX-2 , IL-8 and ICAM-1 mRNA, relative to unstimulated (basal) untransfected cells, were determined by RT-qPCR. Results were normalised to β-actin mRNA expression. Data shows mean ± SD (n = 3). **P<0.01, versus untransfected.

Article Snippet: Proteins were detected using the following antibodies; RCAN1 (D6694; Sigma), NFAT1 (610702; BD Transductions Labs), phospho-p44/42 MAPK (Thr202/Tyr204; #4377), phospho-PKC (pan; #9371), phospho-PLCγ (Tyr 783; #2821), PKCα (#2056), PKCδ (∼2058; all Cell Signalling Technology), PKCε (06-991; Upstate), PKCη (sc-215), colony stimulating factor-1 receptor (CSF-1R; sc-13949) and Actin (sc-1625; all Santa Cruz Biotechnology).

Techniques: Transfection, Knockdown, SDS Page, Western Blot, Control, Expressing, Quantitative RT-PCR