Journal: Cells

Article Title: p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer

doi: 10.3390/cells10123363

Figure Lengend Snippet: Involvement of p38 kinase in resistance to FGFR inhibition. ( A ) Protein expression/phosphorylation levels of FGFR and its direct downstream effectors were analysed with Western blot. Experiments were conducted in triplicates. Representative blots are shown. ( B ) A circos plot showing a copy number variation (relativeLog) in resistant variant of NCI-H1581 (outer circle) and NCI-H1703 (inner circle) cell lines in comparison to respective sensitive variant. Enlarged circos plot with a legible gene names is shown in . ( C ) A heat map showing genes with copy number variation (CNV) between sensitive versus resistant variant (data subjected to rlog transformation). ( D ) A heat map showing differentially expressed genes between sensitive versus resistant cell line variants (only genes with the mutual direction of change in both analysed cell lines are shown; data subjected to rlog transformation).

Article Snippet: To generate cells overexpressing p38 MAPK NCI-H1581 and NCI-H1703 cells were seeded onto 6 cm plates and, after 24 h, transfected with pMT3-p38-HA plasmid (Addgene, #12658) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA).

Techniques: Inhibition, Expressing, Phospho-proteomics, Western Blot, Variant Assay, Comparison, Transformation Assay

Journal: Cells

Article Title: p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer

doi: 10.3390/cells10123363

Figure Lengend Snippet: p38 activity mediates in CPL304110-induced cell growth inhibition. ( A ) Sensitive and resistant variants of NCI-H1581 and NCI-H1703 cells were grown with CPL304110 (0.1 µΜ for NCI-H1581 and NCI-H1581R; 1 µΜ for NCI-H1703 and NCI-H1703R) and/or SB202190 (2 µM) in 3D BD Matrigel ® . Cell growth was measured with ImageJ software after 14 days of culture. Representative pictures were taken. Scale bar represents 100 µm, n = 3. ( B ) Proliferation analysis was evaluated by MTT in sensitive and resistant cells exposed to CPL304110 (0.1 µΜ for NCI-H1581 and NCI-H1581R; 1 µΜ for NCI-H1703 and NCI-H1703R) and/or SB202190 (2 µM) for 96 h. Data are expressed as mean ± SD, ** p ≤ 0.005, n = 3.

Article Snippet: To generate cells overexpressing p38 MAPK NCI-H1581 and NCI-H1703 cells were seeded onto 6 cm plates and, after 24 h, transfected with pMT3-p38-HA plasmid (Addgene, #12658) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA).

Techniques: Activity Assay, Inhibition, Software

Journal: Cells

Article Title: p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer

doi: 10.3390/cells10123363

Figure Lengend Snippet: p38 MAPK overexpression induces resistance to FGFR inhibition. ( A ) p38 kinase overexpression was established in NCI-H1581 and NCI-H1703 cells and confirmed with Western blot. Experiments were conducted in triplicates. Representative blots are shown. ( B ) Cell growth in 3D BD Matrigel ® and ( C ) cell proliferation in the presence of CPL304110 (0.1 µΜ for NCI-H1581, NCI-H1581R, NCI-H1581/p38↑; 1 µΜ for NCI-H1703, NCI-H1703R, NCI-H1703/p38↑) was assessed. Cells were cultured in 3D BD Matrigel ® for 14 days. Representative pictures were taken. Scale bar represents 100 µm, n = 3. Cell proliferation was assessed using MTT viability assay after 96 h. Data are expressed as mean ± SD, *** p ≤ 0.001, n = 3.

Article Snippet: To generate cells overexpressing p38 MAPK NCI-H1581 and NCI-H1703 cells were seeded onto 6 cm plates and, after 24 h, transfected with pMT3-p38-HA plasmid (Addgene, #12658) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA).

Techniques: Over Expression, Inhibition, Western Blot, Cell Culture, MTT Viability Assay

Journal: Journal of Neuroinflammation

Article Title: p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease

doi: 10.1186/s12974-021-02349-y

Figure Lengend Snippet: NLRP3 inflammasome is activated in the α-synucleinA53T-tg mice. A Immunohistochemistry (IHC) demonstrating increased NLRP3 protein levels in the cortex and SNpc of 9–month-old α-synucleinA53T-tg mice. Scale bars, 100 μm. B Statistical analysis of the scores of NLRP3 staining between α-synucleinA53T-tg and wild-type mice. *p < 0.05 (Student’s t-test). C IHC demonstrating increased IL-1β protein levels in the cortex and SNpc of 9–month-old α-synucleinA53T-tg mice. Scale bars, 100 μm. D Statistical analysis of the scores of IL-1β staining between α-synucleinA53T-tg and wild-type mice. *p < 0.05 (Student’s t-test). E – G Cell lysates from the cortex and SNpc of 9-month-old α-synucleinA53T-tg or wild-type mice were immunoblotted. The protein levels of NLRP3, ASC, cleaved CASP1, IL-1β were statistically analyzed in F and G . Mean ± SEM, n = 6, *p < 0.05 (Student’s t-test). H , I IHC demonstrating increased phosphorylated p38 levels in the cortex and SNpc of 9-month-old α-synucleinA53T-tg mice. Scale bars, 100 μm. I Statistical analysis of the scores of phosphorylated p38 between α-synucleinA53T-tg and wild-type mice. *p < 0.05 (Student’s t-test). J , K Lysates from the cortex and SNpc of mice were immunoblotted using the indicated antibodies. The protein levels of phosphorylated p38 and α-synuclein were statistically analyzed in D and E. Mean ± SEM, n = 6, *p < 0.05 (Student’s t-test)

Article Snippet: Plasmids: EGFP-α-synuclein A53T (40823), PHM6-α-synuclein-A53T (40825), pAAV α-synuclein WT (36055), pCDNA3 Flag p38α (20352), and pMT3-p38 (12658) were obtained from Addgene.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Neuroinflammation

Article Title: p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease

doi: 10.1186/s12974-021-02349-y

Figure Lengend Snippet: p38 inhibitor SB203580 inhibits the activation of NLRP3 inflammasome. A IHC demonstrating SB203580 reduced NLRP3 protein levels in the cortex and SNpc of 9-month-old α-synucleinA53T-tg mice. Scale bars, 100 μm. B Statistical analysis of the scores of NLRP3 staining between α-synucleinA53T-tg and wild-type mice. *p < 0.05. C IHC demonstrating SB203580 reduced IL-1β protein levels in the cortex and SNpc of 9-month-old α-synucleinA53T-tg mice. Scale bars, 100 μm. D Statistical analysis of the scores of IL-1β staining between α-synucleinA53T-tg and wild-type mice. *p < 0.05. E – G Cell lysates from the cortex and SNpc of mice were immunoblotted using the indicated antibodies. The protein levels of NLRP3, ASC, cleaved CASP1 were statistically analyzed in F and G . *p < 0.05

Article Snippet: Plasmids: EGFP-α-synuclein A53T (40823), PHM6-α-synuclein-A53T (40825), pAAV α-synuclein WT (36055), pCDNA3 Flag p38α (20352), and pMT3-p38 (12658) were obtained from Addgene.

Techniques: Activation Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease

doi: 10.1186/s12974-021-02349-y

Figure Lengend Snippet: p38 interacts with and phosphorylates TFEB at serine 211. A Lysates from mouse brain were used for IP with anti-TFEB antibody or anti-p38 antibody. B , C Cell lysates from BV2 cells were used for IP with anti-TFEB antibody. SB203580 reduced the TFEB/p38 interaction. *p < 0.05. D , E Cell lysates from BV2 cells were used for IP with anti-p38 antibody. SB203580 reduced the TFEB/p38 interaction and shown in E . Mean ± SEM, n = 3. *p < 0.05. F , G Cell lysates from BV2 cells were used for IP with anti-GFP antibody. SB203580 reduced the TFEB–GFP/14-3-3 interaction. Mean ± SEM, n = 3. *p < 0.05 ( H , I ). Subcellular localization of TFEB was analyzed by confocal microscopy and shown in I. Scale bars, 10 μm. Mean ± SEM, n = 3, *p < 0.05. J , K BV2 cells were labeled with lysosome tracker and visualized lysosome biogenesis under a microscope and shown in K . Scale bars, 100 μm. Mean ± SEM, n = 10, *p < 0.05. L , M Cell lysates from BV2 cells were immunoblotted to determine the levels of NLRP3, ASC, cleaved CASP1, CathB and shown in M . Mean ± SEM, n = 3, *p < 0.05

Article Snippet: Plasmids: EGFP-α-synuclein A53T (40823), PHM6-α-synuclein-A53T (40825), pAAV α-synuclein WT (36055), pCDNA3 Flag p38α (20352), and pMT3-p38 (12658) were obtained from Addgene.

Techniques: Confocal Microscopy, Labeling, Microscopy

Journal: Cell Death & Disease

Article Title: Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T α-synuclein model of Parkinson’s disease

doi: 10.1038/s41419-018-0722-7

Figure Lengend Snippet: a Immunohistochemistry (IHC) staining of phosphorylated p38 in the midbrain of 2, 4, and 6 month α-synuclein A53T -tg or wild-type mice. b Statistical analysis of the average score of phosphorylated p38 staining between α-synuclein A53T -tg and wild-type mice. * p < 0.05 (Student’s t -test). c Cell lysates from primary midbrain cultures and SN4741 cells s were immunoblotted using the indicated antibodies to determine the levels of phosphorylated p38. Mean ± SEM. n = 3. * p < 0.05. ( d , e ) Primary midbrain cultures from wild-type mice and α-synuclein A53T -tg mice. Cultures of both genotype were grown in vitro, fixed, and immunostained for dendritic marker MAP2 and presynaptic marker SYP(synaptophysin) and shown in e as mean ± SEM. n = 3. * p < 0.05. The provided Scale bar in merge image represent 5 μm. Mean ± SEM. n = 3. * p < 0.05

Article Snippet: Other constructs were: EGFP-α-synuclein A53T (Addgene plasmid 40823; Addgene), PHM6-α-synuclein-A53T (Addgene plasmid 40825; Addgene), pAAVα-synuclein WT (Addgene plasmid 36055; Addgene), EGFP-Parkin WT (Addgene plasmid 45875; Addgene), PCDNA3 Flag p38 alpha (Addgene plasmid 20352; Addgene), pMT3-p38 (Addgene plasmid 12658; Addgene).

Techniques: Immunohistochemistry, Staining, In Vitro, Marker

Journal: Cell Death & Disease

Article Title: Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T α-synuclein model of Parkinson’s disease

doi: 10.1038/s41419-018-0722-7

Figure Lengend Snippet: ( a , b ) Mitochondrial membrane potential was assessed by JC-1 and shown in B as mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 50 μm. ( c , d ) Cells transfected with empty vector, α -synuclein A53T were treated with DMSO or SB203580 (10 μM, 24 h). The level of Parkin in the mitochondria component, cytosolic fractions, and total cell lysates were measured by the indicated antibodies. Tublin was used as loading control in total cell lysates and cytosolic fractions, COXIV was used as loading control in mitochondria and shown in ( d ) as mean ± SEM. n = 3. * p < 0.05. ( e , f ) The co-localization analysis of mitochondria and Parkin with mitochondria tracker and anti-Parkin was detected by confocal microscope and presented in ( f ). Mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 5 μm. ( g , h ) The co-localization analysis mitochondria and lysosome with mitochondria tracker and lysosome tracker was examined by confocal microscope and presented in ( h ). Mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 5 μm. i Representative images of Annexin (green) and PI (red) double fluorescence staining showing cell apoptosis. The provided Scale bar in merge image represent 50 μm. j Immunoblotting analyzed the level of apoptotic caspases 3 or poly (ADP-ribose) polymerase and mean ± SEM. n = 3. * p < 0.05

Article Snippet: Other constructs were: EGFP-α-synuclein A53T (Addgene plasmid 40823; Addgene), PHM6-α-synuclein-A53T (Addgene plasmid 40825; Addgene), pAAVα-synuclein WT (Addgene plasmid 36055; Addgene), EGFP-Parkin WT (Addgene plasmid 45875; Addgene), PCDNA3 Flag p38 alpha (Addgene plasmid 20352; Addgene), pMT3-p38 (Addgene plasmid 12658; Addgene).

Techniques: Membrane, Transfection, Plasmid Preparation, Control, Microscopy, Fluorescence, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T α-synuclein model of Parkinson’s disease

doi: 10.1038/s41419-018-0722-7

Figure Lengend Snippet: ( a , b ) Immunohistochemistry (IHC) staining of phosphorylated Parkin of serine 65 in the midbrain of 2, 4, and 6 month α-synuclein A53T -tg or wild-type mice. b Statistical analysis of the average score of phosphorylated Parkin of serine 65 staining between SNCA A53T -tg and wild-type mice. * P < 0.05 (Student’s t -test). ( c , d ) Cell lysates were used for IP with anti-Parkin. Immunoprecipitates or Input were subjected to IB analysis with the indicated antibodies. IgG worked as an immunological control and presented in ( d ). Mean ± SEM. n = 3. * p < 0.05. ( e , f ) SN4741 cells were subjected to imumunofluorescent co-localization analysis with anti-Parkin (green) and anti-PINK1 (red) and detected under confocal microscope and presented in ( f ). Mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 5 μm. ( g , h ) Cell lysates were subjected to immunoblotting analysis with anti-Parkin and phospho-Parkin-ser65 and presented in ( h ). Actin was used as loading control. Mean ± SEM. n = 3. * p < 0.05. ( i , j ) Cell lysates were used for IP with anti-MFN2. Immunoprecipitates or input were subjected to immunoblotting analysis with the indicated antibodies and presented in ( j ). IgG worked as an immunological control. Mean ± SEM. n = 3. * p < 0.05. ( k , l ) Transmission electron microscopy (TEM) was used to observe the mitochondrial morphology. Swollen mitochondria appeared fractured cristae (red arrow) and α-synucleinA53T SN4741 cells treated with SB203580 appeared intact cristae and normal morphological characteristics(blue arrow). The data was presented in ( l ). Mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 2 μm

Article Snippet: Other constructs were: EGFP-α-synuclein A53T (Addgene plasmid 40823; Addgene), PHM6-α-synuclein-A53T (Addgene plasmid 40825; Addgene), pAAVα-synuclein WT (Addgene plasmid 36055; Addgene), EGFP-Parkin WT (Addgene plasmid 45875; Addgene), PCDNA3 Flag p38 alpha (Addgene plasmid 20352; Addgene), pMT3-p38 (Addgene plasmid 12658; Addgene).

Techniques: Immunohistochemistry, Staining, Control, Microscopy, Western Blot, Transmission Assay, Electron Microscopy

Journal: Cell Death & Disease

Article Title: Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T α-synuclein model of Parkinson’s disease

doi: 10.1038/s41419-018-0722-7

Figure Lengend Snippet: ( a , b ) SN4741 cells were transfected with wild-type EGFP-Parkin or its point-mutants (EGFP-Parkin S131A, EGFP-Parkin S136A, EGFP-Parkin S198A, EGFP-Parkin S246A) and p38 MAPK WT. Cell lysates were subjected to IP with anti-GFP antibody, followed by immunoblotting with anti-ser-substrates and anti-GFP antibody, Input were subjected to immunoblotting analysis with anti-GFP antibody and presented in ( b ). Mean ± SEM. n = 3. * p < 0.05. ( c , d ) SN4741 cells transfected with empty vector, α -synuclein WT, α -synuclein A53T were treated with DMSO or SB203580 (10 μM, 24 h). After treatment, the level of Parkin and phospho-Parkin-ser131 were detected by immunoblotting and presented in ( d ). Actin was used as loading control. Mean ± SEM. n = 3. * p < 0.05. ( e – j ) SN4741 cells were transfected with empty vector, α -synuclein A53T, p38 MAPK WT, or kinase-dead p38 MAPK (T182A) in combination. ( e ) The level of Parkin, phospho-Parkin-ser131, were detected by immunoblotting and presented in f . GAPDH was used as loading control. Mean ± SEM. n = 3. * p < 0.05. g The level of Parkin in the mitochondria component, cytosolic fractions, and total cell lysates were measured by the indicated antibodies and presented in h . Tublin was used as loading control in total cell lysates and cytosolic fractions, COXIV was used as loading control in mitochondria. Mean ± SEM. n = 3. * p < 0.05. ( i , j ) The co-localization analysis of mitochondria and lysosome were examined by confocal microscope and showed in j . Mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 5 μm

Article Snippet: Other constructs were: EGFP-α-synuclein A53T (Addgene plasmid 40823; Addgene), PHM6-α-synuclein-A53T (Addgene plasmid 40825; Addgene), pAAVα-synuclein WT (Addgene plasmid 36055; Addgene), EGFP-Parkin WT (Addgene plasmid 45875; Addgene), PCDNA3 Flag p38 alpha (Addgene plasmid 20352; Addgene), pMT3-p38 (Addgene plasmid 12658; Addgene).

Techniques: Transfection, Western Blot, Plasmid Preparation, Control, Microscopy

Journal: Cell Death & Disease

Article Title: Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T α-synuclein model of Parkinson’s disease

doi: 10.1038/s41419-018-0722-7

Figure Lengend Snippet: ( a – h ) SN4741 cells were transfected with EGFP-Parkin S131A and EGFP-Parkin WT together with α -synuclein A53T. ( a ) The co-localization analysis mitochondria and EGFP-Parkin WT or EGFP-Parkin S131A and presented in b . Mean ± SEM. n = 10. * p < 0.05. The provided Scale bar in merge image represent 5 μm. c Cell lysates were used for IP with anti-GFP antibody, immunoblotting detected with anti-PINK1 and anti-GFP antibody, Input were detected with anti-GFP antibody and presented in d . Mean ± SEM. n = 3. * p < 0.05. e Cells lysates were subjected to immunoblotting analysis with Parkin, anti-pSer65Parkin. Tublin was used as loading control. The data was presented in ( f ) and ( g . h ) Cell lysates were used to for IP with anti-MFN2 antibody, immunoblotting detected with anti-UB and anti-MFN2 antibody, Input were detected with anti-MFN2 antibody. The data was presented in i . Mean ± SEM. n = 3. * p < 0.05

Article Snippet: Other constructs were: EGFP-α-synuclein A53T (Addgene plasmid 40823; Addgene), PHM6-α-synuclein-A53T (Addgene plasmid 40825; Addgene), pAAVα-synuclein WT (Addgene plasmid 36055; Addgene), EGFP-Parkin WT (Addgene plasmid 45875; Addgene), PCDNA3 Flag p38 alpha (Addgene plasmid 20352; Addgene), pMT3-p38 (Addgene plasmid 12658; Addgene).

Techniques: Transfection, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Phosphorylation of Parkin at serine 131 by p38 MAPK promotes mitochondrial dysfunction and neuronal death in mutant A53T α-synuclein model of Parkinson’s disease

doi: 10.1038/s41419-018-0722-7

Figure Lengend Snippet: ( a – d ) SN4741 cells and primary midbrain neurons derived from postnatal day 0 mouse pups transfected with empty vector, α-synuclein A53T were treated with SB203580 or DMSO. Cells lysates were subjected to immunoblotting analysis with anti-synaptophysin, anti-synapsin-1, anti-MAP2, and TH. The data presented in c and d , mean ± SEM. n = 3. * p < 0.05. e SN4741 cells were subjected to immunofluorescent staining for presynaptic marker SYP (red) and dendritic marker MAP2 (green) and presented in g . The provided Scale bar in merge image represent 10 μm. Mean ± SEM. n = 10. * p < 0.05. f Primary midbrain neurons derived from postnatal day 0 mouse pups in cultures were subjected to immunofluorescent staining for presynaptic marker SYP (red) and presented in h . The provided Scale bar in merge image represent 5 μm. Mean ± SEM. n = 10. * p < 0.05

Article Snippet: Other constructs were: EGFP-α-synuclein A53T (Addgene plasmid 40823; Addgene), PHM6-α-synuclein-A53T (Addgene plasmid 40825; Addgene), pAAVα-synuclein WT (Addgene plasmid 36055; Addgene), EGFP-Parkin WT (Addgene plasmid 45875; Addgene), PCDNA3 Flag p38 alpha (Addgene plasmid 20352; Addgene), pMT3-p38 (Addgene plasmid 12658; Addgene).

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Western Blot, Staining, Marker

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 5. PEST domain controls the phosphorylation of p38 MAPK. Serum-starved PAE cells expressing VEGFR-2, PEST()VEGFR-2, and PEST()VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and blotted for phospho-p38 (A), total p38 (B), phospho-PLC1 (D), and total PLC1 (E). (C) Quantification of activation of p38. Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and immunoblotted for phospho-p38 (F), total p38 (G), phospho-PLC1 (H), and total PLC1 (I). Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were preincubated with cycloheximide for 90 min, and then cells were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were immunoblotted for VEGFR-2 (J) and total PLC1 (K). (L) Quantification of VEGFR-2 protein levels from blot I is shown. The graph shows averages from two independent experiments. HEK293 cells coexpressing F1173/VEGFR-2 with an empty vector or constitutive active MKK6 (MKK6-Glu) were stimulated with VEGF for the indicated periods of time, and whole-cell lysates were immunoblotted for VEGFR-2 (M), phospho-p38 MAPK (pT180/pY182) (N), p38 MAPK (O), and MKK6 using anti-Flag antibody (P).

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Phospho-proteomics, Expressing, Western Blot, Activation Assay, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 6. MKK6-dependent activation of p38 inhibits downregulation of VEGFR-2. HEK 293 cells coexpressing VEGFR-2 with an empty vector, VEGFR-2 with wild-type p38 MAPK, or VEGFR-2 with dominant-negative p38 (dn-P38) MAPK were preincubated with cycloheximide for 90 min and then stimulated with VEGF for the indicated periods of time. Whole-cell lysates were blotted for VEGFR-2 (A), PLC1 as a control for protein loading (B), and p38 (C). (D) The quantification of the downregulation of VEGFR-2 in response to ligand stimulation. Whole-cell lysates from HEK293 cells coexpressing VEGFR-2 either with an empty vector, constitutive active MKK6 (MKK6-Glu), or with dominant-negative MKK6 (MKK6-Ala) were immunoblotted for VEGFR-2 (E), phospho-VEGFR-2 (pY1054-VEGFR-2) (F), phospho-p38 MAPK (pT180/pY182) (G), p38 MAPK (H), and MKK6 using anti-Flag antibody (I). HEK293 cells expressing VEGFR-2 were transfected either with empty vector or with enhanced green fluorescent protein (EGFP)-tagged Cdc42. Serum-starved cells were stimulated with VEGF for the indicated times, and cells were lysed. Whole-cell lysates were blotted for VEGFR-2 (J), phospo-p38 (K), total p38 (L), and anti-GFP for Cdc42 expression (M). Cells also were treated with cycloheximide (20 mM for 90 min prior to stimulation with VEGF. HUVEC were transfected with control (Ctr.) siRNA or p38 siRNA after 24 h, and cells were starved overnight and stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were blotted with anti-VEGFR-2 antibody (N), anti-Hsp70 antibody (O), and anti-p38 antibody (P). (Q) Ubiquitination of VEGFR-2 in HUVEC in which p38 was silenced. (S) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates were blotted for p38 (R) and Hsp90 (T) for protein loading.

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Activation Assay, Plasmid Preparation, Dominant Negative Mutation, Control, Expressing, Transfection, Ubiquitin Proteomics, Membrane

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 7. p38 is not involved in the ubiquitination of VEGFR-2. HEK293 cells expressing VEGFR-2 were transfected with empty vector, GSK3, dominant-negative p38 (Dn/p38), wild-type p38, dominant-negative PKA catalytic subunit C (Dn/PKA), and wild-type PKA catalytic subunit C [PKA(C)]. Cells were stimulated with VEGF for 10 min, lysed, immunoprecipitated with anti-VEGFR-2 antibody, and blotted with antiubiquitin (Ub; FK2) antibody (A). (B) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates (WCL) from panel A were blotted with anti-phospho-Ser1188 antibody (C), anti-VEGFR-2 antibody (D), anti-GSK3 antibody (E), anti-p38 antibody (F), anti-GFP antibody to detect dominant-negative PKA (G), anti-PKA antibody (H), and anti-PLC1 antibody (I). HEK293 cells expressing VEGFR-2 were transfected with GST-tagged -Trcp1 alone or GST-tagged -Trcp1 with GSK3, -Trcp1 with p38, or -Trcp1 with p38 and GSK3. (J) Cells were stimulated with VEGF for 10 min, immunoprecipitated with anti-VEGFR-2 antibody, and blotted with anti-ubiquitin (FK2) antibody. (K) The same membrane was reblotted for VEGFR-2. (L to N) Whole-cell lysates from panel J were blotted for GSK3, p38, and -Trcp1.

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Plasmid Preparation, Dominant Negative Mutation, Immunoprecipitation, Membrane

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 8. PKA pathway inhibits downregulation of VEGFR-2 and activates p38. PAE cells expressing wild-type VEGFR-2 were pretreated with forskolin (40 M) and then stimulated with VEGF for the indicated time periods. Cells were treated with cycloheximide for 90 min to inhibit protein synthesis. Whole-cell lysates were blotted with anti-VEGFR-2 antibody (A) and with anti-PLC1 antibody for protein loading (B). (C) The quantification of VEGFR-2 downregulation in the presence or absence of forskolin is shown. Serum-starved HEK293 cells coexpressing VEGFR-2 either an empty vector or the catalytic active subunit (C) of PKA were pretreated with cycloheximide (CHX) for 90 min, and then cells were stimulated with VEGF for the indicated time periods. Whole-cell lysates were immunoblotted with anti-VEGFR-2 antibody (D), anti-PLC1 antibody (F), and anti-PKA antibody (G). (E) The quantification of the downregulation of VEGFR-2. The same cell lysates also were blotted for phospho-p38 (H), total p38 (I), phospho-PLC1 (J), total PLC1 (K), phospho-MAPK42/44 (L), and total PKA (M).

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Expressing, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 9. Tyrosine 1173 of VEGFR-2 is required for PKA phosphorylation but not for its association with VEGFR-2. PAE cells expressing VEGFR-2 or F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates (WCL) were blotted for phospho-PKA (A) and total PKA (B). (C) Quantification of the phosphorylation of PKA is shown. It represents averages from two experiments. HEK293 cells were transfected with GFP-tagged PKA catalytic subunit C alone or with Myc-tagged AKAP1. (D) Cells were lysed, immunoprecipitated (Ipt) with anti-GFP antibody, and blotted with anti-Myc antibody. (E) Whole-cell lysates also were blotted for anti-GFP. Serum-starved HEK293 cells coexpressing VEGFR-2 with empty vector or with c-myc-tagged AKAP1 were stimulated with VEGF for the indicated periods of time (F). Cells were lysed, and VEGFR-2 was immunoprecipitated with anti-VEGFR-2 antibody and immunoblotted with anti-c-myc antibody. (G) The same membrane was stripped and reblotted for VEGFR-2. Whole-cell lysates from the same cell groups were immunoblotted for VEGFR-2 (H), total PLC1 (I), phospho-p38 (J), and total p38 (K).

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Phospho-proteomics, Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, Membrane

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 10. Proposed model for PEST-mediated downregulation of VEGFR-2. Upon ligand binding, the PEST domain of VEGFR-2 is phos- phorylated on tyrosine and serine sites, including Y1173, S1188, and Ser1191. Ligand binding promotes -Trcp1 association with VEGFR-2, which mediates the ubiquitination of VEGFR-2 through the Lys-48-dependent ubiquitin chain, leading to degradation by the 26S proteasome system. The activation of PKA by VEGFR-2 requires the AKAP1-mediated association of PKA with VEGFR-2, and its phosphorylation is mediated through Y1173 of VEGFR-2. The activation of PKA leads to the phosphorylation of p38. Activated p38 attenuates the downregulation of VEGFR-2.

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Ligand Binding Assay, Ubiquitin Proteomics, Activation Assay, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 5. PEST domain controls the phosphorylation of p38 MAPK. Serum-starved PAE cells expressing VEGFR-2, PEST()VEGFR-2, and PEST()VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and blotted for phospho-p38 (A), total p38 (B), phospho-PLC1 (D), and total PLC1 (E). (C) Quantification of activation of p38. Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and immunoblotted for phospho-p38 (F), total p38 (G), phospho-PLC1 (H), and total PLC1 (I). Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were preincubated with cycloheximide for 90 min, and then cells were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were immunoblotted for VEGFR-2 (J) and total PLC1 (K). (L) Quantification of VEGFR-2 protein levels from blot I is shown. The graph shows averages from two independent experiments. HEK293 cells coexpressing F1173/VEGFR-2 with an empty vector or constitutive active MKK6 (MKK6-Glu) were stimulated with VEGF for the indicated periods of time, and whole-cell lysates were immunoblotted for VEGFR-2 (M), phospho-p38 MAPK (pT180/pY182) (N), p38 MAPK (O), and MKK6 using anti-Flag antibody (P).

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Phospho-proteomics, Expressing, Western Blot, Activation Assay, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 6. MKK6-dependent activation of p38 inhibits downregulation of VEGFR-2. HEK 293 cells coexpressing VEGFR-2 with an empty vector, VEGFR-2 with wild-type p38 MAPK, or VEGFR-2 with dominant-negative p38 (dn-P38) MAPK were preincubated with cycloheximide for 90 min and then stimulated with VEGF for the indicated periods of time. Whole-cell lysates were blotted for VEGFR-2 (A), PLC1 as a control for protein loading (B), and p38 (C). (D) The quantification of the downregulation of VEGFR-2 in response to ligand stimulation. Whole-cell lysates from HEK293 cells coexpressing VEGFR-2 either with an empty vector, constitutive active MKK6 (MKK6-Glu), or with dominant-negative MKK6 (MKK6-Ala) were immunoblotted for VEGFR-2 (E), phospho-VEGFR-2 (pY1054-VEGFR-2) (F), phospho-p38 MAPK (pT180/pY182) (G), p38 MAPK (H), and MKK6 using anti-Flag antibody (I). HEK293 cells expressing VEGFR-2 were transfected either with empty vector or with enhanced green fluorescent protein (EGFP)-tagged Cdc42. Serum-starved cells were stimulated with VEGF for the indicated times, and cells were lysed. Whole-cell lysates were blotted for VEGFR-2 (J), phospo-p38 (K), total p38 (L), and anti-GFP for Cdc42 expression (M). Cells also were treated with cycloheximide (20 mM for 90 min prior to stimulation with VEGF. HUVEC were transfected with control (Ctr.) siRNA or p38 siRNA after 24 h, and cells were starved overnight and stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were blotted with anti-VEGFR-2 antibody (N), anti-Hsp70 antibody (O), and anti-p38 antibody (P). (Q) Ubiquitination of VEGFR-2 in HUVEC in which p38 was silenced. (S) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates were blotted for p38 (R) and Hsp90 (T) for protein loading.

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Activation Assay, Plasmid Preparation, Dominant Negative Mutation, Control, Expressing, Transfection, Ubiquitin Proteomics, Membrane