antibodies against p mk2  (Cell Signaling Technology Inc)

Journal: The FASEB Journal

Article Title: Phosphorylation‐dependent osterix degradation negatively regulates osteoblast differentiation

doi: 10.1096/fj.202001340r

Figure Lengend Snippet: FIGURE 4 p38 activity negatively regulates abundance and stability of Osx protein (See also Figure S2). A, Immunoblot (IB) analysis of whole-cell lysates (WCLs) derived from UE7T-13. Cells were cultured in osteoblast differentiation medium for 12 hours and serially treated with SB239063 or SB203580 (10 μM) for 24 hours before harvesting. DMSO was used for control cells. B, Real-time RT-PCR analysis to determine the relative mRNA expression levels of Osx in p38 inhibitor-treated UE7T-13 cells presented in (A). Data are presented as mean ± SEM values (n = 3). n.s., not significant; one-way ANOVA with multiple-comparison test. C, IB analysis of WCLs derived from UE7T-13. Cells were cultured in osteoblast differentiation medium for 12 hours, serially treated with SB239063 or SB203580 (10 μM) for 24 hours, and treated with the protein synthesis inhibitor cycloheximide (CHX) (100 μg/mL). The cells were harvested at the indicated time points for IB analysis. Phospho-MK2 is a readout of p38 activity. DMSO was used for control cells. D, Quantification of the relative Osx band intensities in (C), which were normalized to those of the loading control β-actin and to the time point t = 0. Data are presented as mean ± SEM values (n = 3). *P < .05, control versus inhibitor-treated groups; one-way ANOVA with multiple-comparison test

Article Snippet: Anti-p-p38 (9211), anti-p38 (9212), anti-p-Smad (9516), anti-Smad1 (6944), anti-Notch1 (4380), and anti-p-MAPKAPK-2 (MK2) (3007) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Western Blot, Derivative Assay, Cell Culture, Control, Quantitative RT-PCR, Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A temperature-dependent conformational shift in p38α MAPK substrate–binding region associated with changes in substrate phosphorylation profile

doi: 10.1074/jbc.RA119.007525

Figure Lengend Snippet: Temperature dependence of p38 substrate binding is isoform- and substrate-dependent. Unphosphorylated p38α (A and C) or p38β (B and D) were covalently bound to the surface of a CM5 chip, and 0–3 μm MK2 (A and B) or 0–1 μm ATF2 (C and D) were injected at 33, 37, and 39.5 °C. The surface was washed with buffer at the same temperature, and the dissociation of analyte–ligand complexes was followed over time. Because MK:p38 association and dissociation occurred rapidly, KD for p38:MK2 binding was determined using a steady-state affinity model (A and B). The y axis shows response units at equilibrium (Req). The estimated KD, maximal response (Rmax), and the χ2 value for curve fitting are shown for each condition. E, calculated KD values for p38:MK2 and p38:ATF2 binding from SPR analysis at 33, 37, and 39.5 °C. F, melting temperature for p38α, p38β, and ERK2 by DSF; bar graph of 24 measurements each; mean ± S.D.; *, p < 0.0001 versus ERK2; †, p = 0.03 versus p38β.

Article Snippet: Chemicals, recombinant proteins, and antibodies Rabbit anti-phospho-MK2 (Thr-334; catalog no. 3041), rabbit anti- phospho-STAT1 (Ser-727; catalog no. 8826), and rabbit anti-dually phosphorylated (Thr-180/Tyr-182; catalog no. 9215) p38α/β were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Binding Assay, Injection

Journal: The Journal of Biological Chemistry

Article Title: A temperature-dependent conformational shift in p38α MAPK substrate–binding region associated with changes in substrate phosphorylation profile

doi: 10.1074/jbc.RA119.007525

Figure Lengend Snippet: HDX-MS analysis of temperature-dependent conformational changes in p38 MAPK. A, signature EX1 bimodal spectra of peptide segment 130–145 of the p38α p38β (right). The bimodal isotopic envelope (dark blue) was deconvoluted into an exchange-protected envelope (green) or exchange-prone envelope (light blue). B, for segment 130–145, the relative decay of the percentage exchange-protected envelope is plotted for p38α (solid lines) and p38β (dashed line) at 33 °C (turquoise), 37 °C (purple), or 39.5 °C (red) as a function of deuterium incubation time. C, space-filling model of p38α showing spatial relationship of segment 130–145 (αE, red) to the CD (yellow) and ED (brown) motifs and the MK2 C-terminal regulatory domain (orange) lying within the substrate-docking groove of p38α. D, the relative proportion of the percentage exchange-protected envelope after 2 h of deuterium incubation is plotted as a function of temperature for p38α (solid) and p38β (dashed) for segment 207–215 (green), 281–288 (yellow), and 300–306 (blue). E, difference plots are plotted for p38α (top) and p38β (bottom). The differences in percentage deuteration between 39.5 and 33 °C after 10 s (orange), 10 min (blue), and 2 h (purple) deuteration incubation were plotted as a function of the peptide segments from the N to C terminus based on the first residue of the segment. Vertical color bars, peptide segment that displayed EX1 bimodal behavior and are color-coded according to D. Segments with significant differences that do not display EX1 bimodal behavior are denoted by vertical back arrows. F, structural representation of temperature-dependent peptide segments of p38α (top; PDB entry 5UOJ) and corresponding segments from p38β (bottom; PDB entry 3GC8) using the same color coding as in D and E.

Article Snippet: Chemicals, recombinant proteins, and antibodies Rabbit anti-phospho-MK2 (Thr-334; catalog no. 3041), rabbit anti- phospho-STAT1 (Ser-727; catalog no. 8826), and rabbit anti-dually phosphorylated (Thr-180/Tyr-182; catalog no. 9215) p38α/β were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: A temperature-dependent conformational shift in p38α MAPK substrate–binding region associated with changes in substrate phosphorylation profile

doi: 10.1074/jbc.RA119.007525

Figure Lengend Snippet: Temperature-dependent deuteration kinetics for the C-terminal p38α-interacting region of MK2. HDX-MS analysis of MK2 reveals no temperature dependence in the C-terminal p38α-interacting region. A and B, stack spectra of peptidic segment 371–382. The undeuterated control is shown in A. The kinetics of HDX are shown in B at the indicated reaction temperature and deuterium incubation times. The isotopic distribution is shown in red, and the centroid is indicated by the vertical green line. C, relative kinetic deuterium uptake at 33, 37, and 39.5 °C for the peptidic segments 369–382, 371–382, 371–387, and 383–399, covering the entire p38α-interacting region of MK2.

Article Snippet: Chemicals, recombinant proteins, and antibodies Rabbit anti-phospho-MK2 (Thr-334; catalog no. 3041), rabbit anti- phospho-STAT1 (Ser-727; catalog no. 8826), and rabbit anti-dually phosphorylated (Thr-180/Tyr-182; catalog no. 9215) p38α/β were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Incubation