total erk1 2 ab  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: Efficacy and selectivity of the SHP2 inhibitors in cellular cancer models and in patient-derived AML samples. A , viability of AML (Kasumi-1) and esophageal cancer (KYSE-520) cells in the presence of various concentrations of SHP2 active (SBI-2130, SBI-4668, and SBI-3404) and nonactive (SBI6339, negative control) furanylbenzamides after 3 days in culture. Cell viability is shown as a percentage of the vehicle (DMSO) control and represents the mean ± SD (n = 4). B , SBI-4668 dose–response curves in cell viability assays using Kasumi-1, KYSE-520, MOLM-13, and MV4-1 cells after 3 days in culture. The percentages compared with DMSO vehicle control were curve fitted using nonlinear regression (log[inhibitor] versus normalized response, variable slope) and represent the mean ± SD (n = 4). C , cell viability of BT-459 and MDA-MB-468 triple-negative breast cancer (TNBC) cells in the presence of various concentrations of SBI-4668 or the SHP2 allosteric inhibitor SHP099 after 5 days in culture. Cell viability is shown as a percentage of DMSO vehicle control, representing mean ± SD (n = 2), and curve fitted as in B . D , colony formation assay (11 days) of BT-459 and MDA-MB-468 TNBC cells in the presence of various concentrations of SBI-4668 or the SHP2 allosteric inhibitor SHP099. E , phospho-ERK1/2 (p-ERK1/2) immunoblot analysis from total cell lysates of MOLM-13 AML cells treated with SBI-4668 at the indicated concentrations or with SHP2 allosteric inhibitor RMC-4550 (RMC, 1 μM) for 3 h or 24 h. The quantitation of p-ERK1/2 levels is shown as the percentage of the DMSO (vehicle) control and represents data from three independent experiments (mean ± SD). F , viability of AML patient-derived cells in the presence of 10 μM SBI-4668 or 10 μM SHP2 allosteric inhibitor RMC-4550 after 2, 4, or 6 days in culture. Cell viability is shown as a percentage of the DMSO vehicle control and represents the mean ± SD (n = 4; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001; unpaired t test with Welch's correction). G , viability of MOLM-13 cells and MOLM-13-Cas9-mCherry cells with SHP2 KO in the presence of SBI-2130, SBI-4668, and allosteric inhibitor SHP099 at various concentrations (10-point dose response). The percentages compared with the DMSO vehicle control were curve fitted using nonlinear regression (log[inhibitor] versus normalized response, variable slope) and represent the mean ± SD (n = 4). SHP2 protein levels in regular MOLM-13 cells (WT), MOLM-13-Cas9-mCherry cells (Cas9), and MOLM-13-Cas9-mCherry cells with SHP2 KO were evaluated by immunoblot analysis using SHP2 antibodies. AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; SHP2, Src-homology 2 domain–containing phosphatase 2.

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques: Derivative Assay, Negative Control, Colony Assay, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: Evaluation of SBI-4668 in U-937 AML cells expressing the SHP2 oncogenic variant G60R. A , cell viability of U-937 AML cells in the presence of various concentrations of SBI-4668 or the SHP2 allosteric inhibitor RMC-4550 after 3 days in culture. Cell viability is shown as a percentage of the DMSO vehicle control, representing the mean ± SD (n = 4) and curve fitted using nonlinear regression (log[inhibitor] versus normalized response, variable slope). B , phospho-ERK1/2 (p-ERK1/2) immunoblot analysis from total cell lysates of U-937 AML cells treated with SBI-4668 at the indicated concentrations or with SHP2 allosteric inhibitor RMC-4550 (RMC, 1 μM) for 24 h. The quantitation of p-ERK1/2 levels is shown as the percentage of the DMSO (vehicle) control and represents data from three independent experiments (mean ± SD). AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; SHP2, Src-homology 2 dom/ain–containing phosphatase 2.

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques: Expressing, Variant Assay, Western Blot, Quantitation Assay

Journal: Journal of Lipid and Atherosclerosis

Article Title: Curcumin Attenuates Acrolein-induced COX-2 Expression and Prostaglandin Production in Human Umbilical Vein Endothelial Cells

doi: 10.12997/jla.2020.9.1.184

Figure Lengend Snippet: Cells were pre-treated with curcumin (10 and 25 µM) for 30 minutes prior to acrolein treatment (10 µM). After 30 minutes of incubation, the cell lysates (20 µg) were prepared and western blot analysis was performed with Abs against phosphorylated PKCδ, p38, and CREB or total PKCδ, p38, and CREB. Quantitative data were obtained using an imaging densitometer (ImageJ version 1.52a software, NIH, Bethesda, MD, USA). Data were analyzed by the Student's t -test. Data represent the mean±standard deviation of results from 3 independent experiments. PKC, protein kinase C; CREB, cAMP response element-binding protein; HUVEC, human umbilical vein endothelial cell; Ab, antibody; p, phosphorylated. * p <0.05 vs. control group.

Article Snippet: Abs against phospho-specific p38 MAPK, protein kinase C (PKC) δ, CREB, and Abs against total p38 MAPK, PKCδ, and CREB were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Techniques: Incubation, Western Blot, Imaging, Software, Standard Deviation, Binding Assay, Control

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Neurotoxic CXCL12 transiently activates p38 MAPK and JNK in rat cerebrocortical cultures. a Complete neuro-glial cell cultures and neuron-depleted cerebrocortical cell cultures were analyzed by Western blotting for the presence of active p38 MAPK and JNK. To obtain glial cells, neurons were depleted by treating cerebrocortical cultures with 300 μM NMDA for 20 min 2 days prior to the preparation of cell lysates. Equal amounts of cellular protein (30 μg) were separated by SDS-PAGE and analyzed by immunoblotting for the indicated proteins. A representative Western blot from one of the three independent neuron depletion experiments is shown. b Cerebrocortical cultures were incubated with recombinant CXCL12 (20 nM) for the indicated time periods, prior to cell lysis on ice. Equal amounts of cellular protein (100 μg) were used for the performance of immunocomplex kinase assays as described in the “ ” section. The Western blots show representative samples of phosphorylated indicator substrates observed with samples of the 12 and 24 h time points. Kinase activity in CXCL12-exposed samples and vehicle-treated controls was measured for each time point. Vehicle controls were defined as the 100 % baseline value. Note the difference in the dimension of time on the split X -axis. Each time point represents four to seven assessments in duplicate or triplicate in independent experiments. * P ≤ 0.05 compared to control by student’s t test. ‘pp38’/‘p-p38’ and ‘p-JNK’ indicate phosphorylated p38 MAPK and JNK, respectively

Article Snippet: After blocking remaining binding sites with BSA, the membranes were probed sequentially with anti-phospho p38 MAPK Ab (Promega, WI), total p38 MAPK Ab (Cell Signaling Technologies), anti-phospho c-Jun N-terminal kinase (JNK) Ab, total JNK Ab (both Cell Signaling Technologies), anti-NeuN Ab (Chemicon), anti-synaptophysin Ab (Dako), anti Actin Ab (Millipore), and anti-α-tubulin Ab (Sigma) as described earlier [ , ].

Techniques: Western Blot, SDS Page, Incubation, Recombinant, Lysis, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Phosphorylated p38 MAPK in cerebrocortical cell cultures localizes to neurons with and without CXCL12 exposure. Cerebrocortical cell cultures from rats were incubated with CXCL12 (20 nM). After 12 h of treatment, cells were fixed, permeabilized, and stained for MAP-2 ( red ), as a neuronal marker, activated-phospho p38 MAPK ( green ), and nuclear DNA (DRAQ5; shown in blue pseudocolor for better visualization). Samples were analyzed using immunofluorescence microscopy as described in the “ ” section. The overlap of red and green signals appears yellow in the merged images. Scale bars , 20 μm

Article Snippet: After blocking remaining binding sites with BSA, the membranes were probed sequentially with anti-phospho p38 MAPK Ab (Promega, WI), total p38 MAPK Ab (Cell Signaling Technologies), anti-phospho c-Jun N-terminal kinase (JNK) Ab, total JNK Ab (both Cell Signaling Technologies), anti-NeuN Ab (Chemicon), anti-synaptophysin Ab (Dako), anti Actin Ab (Millipore), and anti-α-tubulin Ab (Sigma) as described earlier [ , ].

Techniques: Incubation, Staining, Marker, Immunofluorescence, Microscopy

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Ca 2+ channel blockers limit activity of p38 MAPK in cerebrocortical cells upon exposure to CXCL12. Rat cerebrocortical cells were incubated for the indicated time periods with CXCL12 (20 nM) in the presence or absence of MK-801 (10 μM) or nimodipine (10 nM), prior to cell lysis on ice. 30 μg of protein per lane were analyzed by Western blotting using the specific antibodies against the indicated molecules. A representative immunoblot is shown for each time point. Quantification by densitometry; n = 3 to 4 per time point; * P ≤ 0.05, ** P ≤ 0.01 by ANOVA followed by Fisher’s PLSD post hoc test

Article Snippet: After blocking remaining binding sites with BSA, the membranes were probed sequentially with anti-phospho p38 MAPK Ab (Promega, WI), total p38 MAPK Ab (Cell Signaling Technologies), anti-phospho c-Jun N-terminal kinase (JNK) Ab, total JNK Ab (both Cell Signaling Technologies), anti-NeuN Ab (Chemicon), anti-synaptophysin Ab (Dako), anti Actin Ab (Millipore), and anti-α-tubulin Ab (Sigma) as described earlier [ , ].

Techniques: Activity Assay, Incubation, Lysis, Western Blot

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Phosphorylated p38 MAPK localization in neurons in the presence of CXCL12 and Ca 2+ channel blockers. Cerebrocortical cell cultures were exposed to CXCL12 (20 nM), nimodipine (10 nM), or MK801 (10 μM) or combinations thereof for 12 h. Afterwards, cells were fixed, permeabilized, and stained for MAP-2 ( red ), activated/phospho p38 MAPK ( green ) and nuclear DNA (DRAQ5; shown in blue pseudocolor for better visualization). Images were analyzed using fluorescence microscopy as described in the “ ” section. The overlap of red and green signals appears yellow in the merged images. Scale bars , 20 μm

Article Snippet: After blocking remaining binding sites with BSA, the membranes were probed sequentially with anti-phospho p38 MAPK Ab (Promega, WI), total p38 MAPK Ab (Cell Signaling Technologies), anti-phospho c-Jun N-terminal kinase (JNK) Ab, total JNK Ab (both Cell Signaling Technologies), anti-NeuN Ab (Chemicon), anti-synaptophysin Ab (Dako), anti Actin Ab (Millipore), and anti-α-tubulin Ab (Sigma) as described earlier [ , ].

Techniques: Staining, Fluorescence, Microscopy

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Pharmacological inhibition of p38 MAPK protects cerebrocortical neurons from toxicity of CXCL12. Cerebrocortical cell cultures were incubated with CXCL12 (20 nM) in the presence and absence of p38 MAPK inhibitor SB203580 (SB, 10 μM) for 24 h. Analysis of neuronal survival was performed as described in the “ ” section using staining for neurons (MAP-2) and nuclear DNA (H33342). Values are mean ± SEM; n ≥ 3 with duplicate or triplicate samples per condition; ** P ≤ 0.01, *** P ≤ 0.001 by ANOVA followed by Fisher’s PLSD post hoc test

Article Snippet: After blocking remaining binding sites with BSA, the membranes were probed sequentially with anti-phospho p38 MAPK Ab (Promega, WI), total p38 MAPK Ab (Cell Signaling Technologies), anti-phospho c-Jun N-terminal kinase (JNK) Ab, total JNK Ab (both Cell Signaling Technologies), anti-NeuN Ab (Chemicon), anti-synaptophysin Ab (Dako), anti Actin Ab (Millipore), and anti-α-tubulin Ab (Sigma) as described earlier [ , ].

Techniques: Inhibition, Incubation, Staining

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: NMDA receptors and l -type Ca 2+ channels can regulate CXCL12 induced neuronal death upstream of p38 MAPK activation. Activity of p38 MAPK (phosphorylation indicated by p ) increases above baseline levels ( upward arrow ) as a critical mediator of CXCR4-mediated neurotoxicity of CXCL12. The present study implicates besides NMDAR-gated ion channels for the first-time l -type Ca 2+ channels ( l -type CC) in CXCL12 neurotoxicity as the blockade of both independently can prevent CXCL12 neurotoxicity while concomitantly preventing an increase (nimodipine; equal sign ) or reducing (MK-801; downward arrow) the activity of p38 MAPK compared to baseline

Article Snippet: After blocking remaining binding sites with BSA, the membranes were probed sequentially with anti-phospho p38 MAPK Ab (Promega, WI), total p38 MAPK Ab (Cell Signaling Technologies), anti-phospho c-Jun N-terminal kinase (JNK) Ab, total JNK Ab (both Cell Signaling Technologies), anti-NeuN Ab (Chemicon), anti-synaptophysin Ab (Dako), anti Actin Ab (Millipore), and anti-α-tubulin Ab (Sigma) as described earlier [ , ].

Techniques: Activation Assay, Activity Assay

Journal: Acta Neuropathologica

Article Title: Soluble VCAM-1 impairs human brain endothelial barrier integrity via integrin α-4-transduced outside-in signalling

doi: 10.1007/s00401-015-1417-0

Figure Lengend Snippet: Soluble VCAM-1 activates Rho GTPase and p38 MAP kinase in brain endothelial cells. a Western blot analysis of Rho activation in integrin α-4-positive primary HBMEC after stimulation with 5 µg/mL sVCAM-1 for 10 min. b Western blot analysis of p38 MAP kinase activation after stimulation with 5 µg/mL sVCAM for the indicated durations. Stimulation with 12- O -tetradecanoylphorbol-13-acetate (TPA) and ionomycin for 10 min was used as a positive control. Representative of four independent experiments with different primary cell preparations

Article Snippet: After peroxidase inactivation, membranes were reprobed with Abs against total p38 (cat.-no. 9212, Cell Signaling), ERK (cat.-no. sc-94, Santa Cruz) or JNK (cat.-no. sc-474, Santa Cruz).

Techniques: Western Blot, Activation Assay, Positive Control

Journal: Acta Neuropathologica

Article Title: Soluble VCAM-1 impairs human brain endothelial barrier integrity via integrin α-4-transduced outside-in signalling

doi: 10.1007/s00401-015-1417-0

Figure Lengend Snippet: Effects of sVCAM-1 on tight junction morphology are partially mediated by Rho GTPase and p38 MAP kinase activation. Immunocytochemical analysis of F-actin ( a ) and occludin ( b ) expression in primary HBMEC which were left untreated, treated with 2 µM Rho-associated kinases inhibitor Y-27632 or 10 µM p38 inhibitor SB203580 for 2 h, 5 µg/mL sVCAM-1 for 1 h, or by one of the inhibitors for 2 h and additional presence of 5 µg/mL sVCAM-1 for the last hour. Representative of five independent experiments evaluated by blinded investigators. Scale bar 25 µm

Article Snippet: After peroxidase inactivation, membranes were reprobed with Abs against total p38 (cat.-no. 9212, Cell Signaling), ERK (cat.-no. sc-94, Santa Cruz) or JNK (cat.-no. sc-474, Santa Cruz).

Techniques: Activation Assay, Expressing