Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Tissue factor Deficiency Increases Alveolar Hemorrhage and Death in Influenza A Infected Mice

doi: 10.1111/jth.13307

Figure Lengend Snippet: We analyzed TF antigen expression in the lungs of uninfected (B, E) and infected (7 dpi) wild-type mice (C, F), and infected TFΔEp mice (G–I) by immunohistochemistry. Tissue sections were incubated with (B, C, E, F–I) or without (A and D) a goat anti-mouse TF polyclonal antibody. The black arrows indicate TF expression in the epithelium of bronchi (Bc). The arrowheads indicate TF staining in adventitial cells of blood vessels (Bv). The white arrow indicates TF expression in adventitial cells surrounding a bronchiole. Original magnification x200. Scale bar is 100 µm.

Article Snippet: Then, slides were incubated overnight at 4°C with 1 μg/mL anti-human TF goat polyclonal antibody (R&D Systems, Minneapolis, MN), rinsed and incubated with 1.5 μg/mL biotinylated rabbit anti-goat secondary antibody (Vector Lab) for 20 min at room temperature.

Techniques: Expressing, Infection, Immunohistochemistry, Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Analysis of PP5-ERK interactions and PP5 activity. A, HEK-293FT cells were transfected with FLAG-PP5, HA-ERK1, or HA-ERK2 alone or in combination, as indicated. Western analysis of FLAG immune complexes (FLAG IPs), HA immune complexes (HA IPs), and cell lysates were performed using FLAG, HA, and HSP90 antibodies. *, IgG heavy chain. B, HEK-293FT cells were transfected with FLAG-PP5, HA-ERK1b, or HA-ERK1c alone or in combination, as indicated. FLAG IPs and cell lysates were analyzed by Western as in A. C, HEK-293FT cells were transfected with wild type FLAG-PP5 or FLAG-PP5HBD alone or together with HA-ERK1 or HA-ERK2; cells were also transfected with the indicated HA-tagged kinase and pcDNA3 (Vector). FLAG IPs and cell lysates were analyzed as described in A. A significant reduction in the binding of HA-ERK1 (88.21 ± 3.01%, p < 0.0001) and HA-ERK2 (77.3 ± 8.97%, p = 0.0003) to FLAG-PP5HBD was found when ERK signals were normalized to levels of mutant PP5 in the IPs and compared with the corresponding values in the wild type FLAG-PP5 conditions, which were set to 100. The results represent the means ± S.E. analyzed by one-sample t test using two-tailed p values. D, approximately 118 ng (10 nm) of purified wild type FLAG-PP5 (WT) or HSP90 binding-deficient mutant of FLAG-PP5 (HBD) were continuously assayed over 900 s for phosphatase activity toward DiFMUP (relative fluorescent units (RFU)) in the presence of only buffer (Basal), 100 μm arachidonic acid (AA), or 200 nm S100B plus 1 mm CaCl2 (S100B). Background fluorescence (i.e. samples containing only DiFMUP + arachidonic acid or DiFMUP + S100B + CaCl2) was measured and subtracted from the corresponding fluorescent values of the phosphatase-containing samples. Levels of fluorescence in WT + arachidonic acid, HBD + arachidonic acid, and HBD + S100B preparations were virtually identical. The results represent the means ± S.E. from six independent experiments, three experiments performed with duplicates from each of two separate purifications of WT and HBD. S.E. bars are obscured by the symbols for most data points. E, quantification of phosphatase activity at the 900 s time point. Two-way analysis of variance identified a statistically significant genotype versus activator interaction (F(5,30) = 61.83, p < 0.0001). Tukey post-tests are shown as follows: ***, versus basal, p < 0.0001; ^^^, WT versus HBD, p < 0.0001. Error bars, S.E. F, HEK-293FT cells expressing FLAG-PP5 and HA-ERK2 were lysed in Buffer B (B) or RIPA buffer (R) (Lysates). FLAG immunoprecipitations (FLAG IPs) were performed from the cell lysates and washed (IP Wash) in either Buffer B or RIPA buffer, as indicated. The FLAG IPs and corresponding cell lysates were analyzed by Western using HSP90, HA, and FLAG antibodies. The data are representative of experiments performed three (A), three (B), six (C), six (D), and two (F) independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Activity Assay, Transfection, Western Blot, Plasmid Preparation, Binding Assay, Mutagenesis, Two Tailed Test, Purification, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: The interaction of PP5 with ERK1/2 is independent of kinase and phosphatase activity. A, FLAG immunoprecipitations (FLAG IPs) were performed from lysates of HEK-293FT cells transfected with wild type FLAG-PP5, kinase-dead ERK1 (HA-ERK1KD), or kinase-dead ERK2 (HA-ERK2KD) alone or in combination, as indicated. The cell lysates and FLAG IPs were subjected to Western analysis using FLAG, HA, and HSP90 antibodies. B, FLAG IPs were performed from lysates of HEK-293FT cells transfected with phosphatase-dead PP5 (FLAG-PP5PD), wild type HA-ERK1, or wild type HA-ERK2 alone or in combination, as indicated. The cell lysates and FLAG IPs were analyzed as described in A. *, IgG heavy chain. The data are representative of experiments performed three (A) and three (B) independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Activity Assay, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Active Rac1 promotes assembly of PP5·ERK1 and PP5·ERK2 complexes. A, HEK-293FT cells were co-transfected with FLAG-PP5 and either HA-ERK1 or HA-ERK2 together with pcDNA3 (Vector), constitutively active Rac1 (Rac1L61), or dominant negative Rac1 (Myc-Rac1N17), as indicated. Western analysis of FLAG immune complexes (FLAG IPs) and cell lysates were performed using the HA, FLAG, HSP90, and Rac1 antibodies. B, quantification of the percentage of maximal HA-ERK binding normalized to the FLAG-PP5 signal in FLAG IPs, with binding in the vector samples set to 100. One-way analysis of variance identified a significant increase in PP5-ERK1 (F(2,6) = 6.222, p = 0.0344) and PP5-ERK2 (F(2,6) = 17.28, p = 0.0032) association in the presence of Rac1L61. Tukey post-tests are shown as follows: *, p < 0.05; **, p < 0.01. Data are mean ± S.E. No significant differences in the expression levels of FLAG-PP5, HA-ERK1, or HA-ERK2 were detected following normalization to HSP90 levels. C, HEK-293FT cells were transfected with pcDNA3 (Vector) or FLAG-PP5 in the absence (−) or presence (+) of constitutively active Rac1 (Rac1L61). Cells transfected with Rac1L61 were also treated with 100 ng/ml EGF for 5 min prior to lysis. Endogenous ERK1/2 immune complexes (ERK1/2 IPs) and cell lysates were analyzed by Western using phospho-ERK1/2 (p-ERK1/2), ERK2, PP5, and FLAG antibodies. D, HEK-293FT cells were co-transfected with HA-ERK2 and pcDNA3 (−) or Rac1L61 (+). Western analysis of cell lysates and proteins purifying with normal rabbit IgG (IgG IPs), rabbit anti-PP5 antibody (PP5 IPs), and microcystin-agarose (MC PDs) were performed using ERK2 and PP5 antibodies. E, lysates from untransfected (left) and HA-ERK2-expressing (right) HEK-293FT cells were incubated with microcystin-agarose, and bound proteins were extensively washed prior to splitting the resin into separate tubes, which were then incubated with buffer lacking (−) or containing (+; 20 μg) purified S100A1. A fraction of the reaction mixture was collected and analyzed by SDS-PAGE (15% Tris-glycine gels), and stained with Coomassie G-250 to detect S100A1. Following incubation, bound proteins were extensively washed and eluted for analysis by Western blotting with antibodies detecting the ERK1/2 and PP5 proteins. Unpaired, one-tailed t tests identified a significant decrease in the levels of bound ERK following incubation with S100A1 (left, *, p = 0.0117; right, *, p = 0.0279). Error bars, S.E. The data are representative of experiments performed three (A), three (C), two (D), three (E, left), and two (E, right) independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Binding Assay, Expressing, Lysis, Incubation, Purification, SDS Page, Staining, One-tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Specific oncogenic Ras variants selectively decrease PP5-ERK2, but not PP5-ERK1, interactions. A, HEK-293FT cells were transfected with HA-ERK1, HA-ERK2, or FLAG-PP5 alone or in combination and treated with nothing (Ø), 50 ng/ml EGF (5 min), 100 nm PMA (20 min), or an equivalent volume of DMSO (20 min) as a vehicle control. HEK-293FT cells transfected with FLAG-PP5 and HA-ERK1 or HA-ERK2 in combination with pcDNA3 (Vector) or HRasV12 were not treated prior to lysis. Western analysis of the FLAG immune complexes (FLAG IPs) and cell lysates was done using antibodies recognizing phospho-ERK1/2 (p-ERK1/2), Ras, HSP90, HA, and FLAG. Note that phospho-HA-ERK2 (p-HA-ERK2) and endogenous phospho-ERK1 (endog. p-ERK1) co-migrate. B, HEK-293FT cells transfected with (+) or without (−) HA-ERK2 or FLAG-PP5 were co-transfected with HRasV12, wild type HRas (HRasWT), HA-KRasV12, HA-KRasL61, or wild type HA-KRas (HA-KRasWT). FLAG IPs and cell lysates were analyzed by Western blot as in A. The data are representative of experiments performed four (A) and three (B) independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Transfection, Plasmid Preparation, Lysis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: Kinase activity, but not phosphatase activity, is required for HRasV12-dependent disruption of the PP5·ERK2 complex. A, HEK-293FT cells were transfected with wild type HA-ERK1 or wild type HA-ERK2 together with (+) or without (−) phosphatase-dead FLAG-PP5 (FLAG-PP5PD) and HRasV12. FLAG immune complexes (FLAG IPs) and cell lysates were analyzed by Western blotting using antibodies recognizing the indicated proteins. B, wild type HA-ERK1 (HA-ERK1WT), kinase-dead HA-ERK1 (HA-ERK1KD), wild type HA-ERK2 (HA-ERK2WT), and kinase-dead HA-ERK2 (HA-ERK2KD) were transfected into HEK-293FT cells alone or together with wild type FLAG-PP5 in the absence or presence of HRasV12. FLAG IPs and cell lysates were analyzed as described in A. *, IgG heavy chain. The data are representative of experiments performed three (A) and five (B) independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Activity Assay, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: HRasV12 induces disruption of the PP5·ERK2 complex independently of the activation state of ERK2. A, HEK-293FT cells were transfected with (+) or without (−) HA-ERK2 and FLAG-PP5 together with HRasV12 or wild type HRas (HRasWT); cells were treated with the MEK inhibitor U0126 or DMSO for 30 min prior to lysis. FLAG immune complexes (FLAG IPs) and cell lysates were analyzed by Western using antibodies recognizing the indicated proteins. B, percentage of maximal binding of HA-ERK2 to FLAG-PP5. ERK2 binding signals, quantified for cells in A that co-expressed HA-ERK2 and FLAG-PP5, were normalized to levels of PP5 in the FLAG IPs and compared with the corresponding values in the absence of any Ras expression (Ø), which were set to 100. Significant reductions in PP5 binding to ERK2 were observed in the presence of HRasV12 (86.08 ± 1.62%) and following acute treatment with U0126 (81.95 ± 2.26%), whereas HRasWT expression failed to disrupt the interaction. The results represent the means ± S.E. based on one-way analysis of variance (F(3,8) = 20.42, p = 0.0004). Tukey post-tests are shown as follows: **, versus Ø, p < 0.01; ##, versus HRasWT, p < 0.01. Error bars, S.E. The data are representative of experiments performed three independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Activation Assay, Transfection, Lysis, Western Blot, Binding Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: PP5·ERK2 complexes regulate Raf1 feedback phosphorylation, which is elevated in Rac1L61- and HRasV12-expressing cells. A, HEK-293FT cells were co-transfected with Myc-Raf1 or pcDNA3 (EV) together with the indicated combinations of nothing (Ø), wild type (WT) or kinase-dead HA-ERK2 (KD), and wild type (WT) or catalytically inactive FLAG-PP5 (PD). Cells were treated with solvent containing (+) or lacking (−) 100 ng/ml EGF for 30 min prior to lysis. FLAG immune complexes (FLAG IPs) were analyzed by Western using phospho-Ser-289/296/301-Raf1 (pEDS-Raf1), phospho-Ser-338-Raf1 (p338-Raf1), Raf1, HSP90, and FLAG antibodies. B, HEK-293FT cells were co-transfected to express Myc-Raf1, in the presence or absence of Rac1L61 or HRasV12, together with the indicated combinations of nothing (Ø), wild type (WT) or kinase-dead HA-ERK2 (KD), and wild type (WT) or catalytically inactive FLAG-PP5 (PD). FLAG IPs were analyzed as in A. The data are representative of experiments performed two (A) and two (B) independent times with similar results.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Expressing, Transfection, Lysis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1 *

doi: 10.1074/jbc.M113.518514

Figure Lengend Snippet: A model depicting the role and regulation of PP5·ERK complexes. PP5 suppresses Raf1 signaling by dephosphorylating Ser-338, a site important in making Raf1 permissive to further phosphorylation for full activation. Our studies support an additional role for PP5 in regulating the phosphorylation state of several EDS on Raf1 and suggest that PP5·ERK complexes coordinate Raf1 feedback phosphorylation events. Furthermore, we find that PP5-ERK interactions are modulated by active small G proteins. Active Rac1 (Rac1L61) promotes PP5-ERK1/2 interactions. In contrast, active HRas (HRasV12) and KRas4B (KRasL61), but not KRasV12, promote rapid turnover of PP5·ERK2 complexes without affecting PP5·ERK1 complexes.

Article Snippet: The mouse anti-PP5 and mouse anti-Raf1 antibodies were from BD Biosciences.

Techniques: Activation Assay