Structured Review

Santa Cruz Biotechnology anti phospho erk
Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The <t>4G10</t> (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho <t>ERK</t> and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.
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1) Product Images from "Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML"

Article Title: Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML

Journal: Oncogene

doi: 10.1038/onc.2016.41

Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.
Figure Legend Snippet: Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

Techniques Used: Concentration Assay, Cell Culture, Immunoprecipitation, SDS Page, Western Blot

2) Product Images from "MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts"

Article Title: MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg322

Fig. 7. Fibroblast migration and calpain activity are dependent on MEK, but not JNK activity. ( A ) Wild-type fibroblasts were loaded into Transwell migration chambers (10 5 cells/well) and allowed to migrate for 5 h, using 5% serum as a chemotactic agent. Calpain inhibitor PD150606 (50 µM), MEK inhibitor UO126 (10 µM) or matrix metalloproteinase inhibitor GM6001 (2 µM) were added to both the upper and lower chambers of the designated wells. ( B ) Wild-type fibroblasts were pre-incubated for 1 h with JNK inhibitor SP600125 (10 µM) or MEK inhibitor UO126 (10 µM), and calpain activity was assessed by SLLVY-AMC cleavage, and compared with that of non-treated cells. The results of both (A) and (B) are the mean ± SEM of at least three independent experiments. ( C ) Wild-type fibroblasts were pre-incubated with 10 µM SP600125 or 10 µM UO126 for 1 h and then analyzed for migration using the in vitro wound healing assay in the continuous presence of inhibitor. ( D ) Serum-starved wild-type, MEKK1–/– or MEKK1 add-back fibroblasts were treated with EGF or FGF-2 for 10 min and then lysed. ERK1/2 activation was then assessed by phospho-ERK immunoblotting. The membrane was then stripped and the total ERK2 level determined by ERK2 immunoblotting. The data are representative of at least three independent experiments. NS, no stimulus.
Figure Legend Snippet: Fig. 7. Fibroblast migration and calpain activity are dependent on MEK, but not JNK activity. ( A ) Wild-type fibroblasts were loaded into Transwell migration chambers (10 5 cells/well) and allowed to migrate for 5 h, using 5% serum as a chemotactic agent. Calpain inhibitor PD150606 (50 µM), MEK inhibitor UO126 (10 µM) or matrix metalloproteinase inhibitor GM6001 (2 µM) were added to both the upper and lower chambers of the designated wells. ( B ) Wild-type fibroblasts were pre-incubated for 1 h with JNK inhibitor SP600125 (10 µM) or MEK inhibitor UO126 (10 µM), and calpain activity was assessed by SLLVY-AMC cleavage, and compared with that of non-treated cells. The results of both (A) and (B) are the mean ± SEM of at least three independent experiments. ( C ) Wild-type fibroblasts were pre-incubated with 10 µM SP600125 or 10 µM UO126 for 1 h and then analyzed for migration using the in vitro wound healing assay in the continuous presence of inhibitor. ( D ) Serum-starved wild-type, MEKK1–/– or MEKK1 add-back fibroblasts were treated with EGF or FGF-2 for 10 min and then lysed. ERK1/2 activation was then assessed by phospho-ERK immunoblotting. The membrane was then stripped and the total ERK2 level determined by ERK2 immunoblotting. The data are representative of at least three independent experiments. NS, no stimulus.

Techniques Used: Migration, Activity Assay, Incubation, In Vitro, Wound Healing Assay, Activation Assay

3) Product Images from "NGF Accelerates Cutaneous Wound Healing by Promoting the Migration of Dermal Fibroblasts via the PI3K/Akt-Rac1-JNK and ERK Pathways"

Article Title: NGF Accelerates Cutaneous Wound Healing by Promoting the Migration of Dermal Fibroblasts via the PI3K/Akt-Rac1-JNK and ERK Pathways

Journal: BioMed Research International

doi: 10.1155/2014/547187

The effect on human skin fibroblast migration of blocking JNK, ERK, or PI3K/Akt pathways with specific inhibitors. ((A1), (B1), and (C1)) The levels of Akt, JNK, and ERK activity were all enhanced after treatment with 100 ng/mL of NGF for 5, 15, or 30 min. ((A2), (B2), and (C2)) The activity of Akt, JNK, or ERK following 5 min of NGF treatment was abolished by the corresponding specific inhibitor: LY294002 (10 μ M, LY is the abbreviation for LY294002), SP600125 (10 μ M, SP is the abbreviation for SP600125), or PD98059 (10 μ M, PD is the abbreviation for PD98059). ((A3), (A4), (B3), (B4), (C3), and (C4)) The NGF-induced migration of human skin fibroblasts induced by NG was significantly impaired after incubation with LY294002 (10 μ M), SP600125 (10 μ M), or PD98059 (10 μ M). * P
Figure Legend Snippet: The effect on human skin fibroblast migration of blocking JNK, ERK, or PI3K/Akt pathways with specific inhibitors. ((A1), (B1), and (C1)) The levels of Akt, JNK, and ERK activity were all enhanced after treatment with 100 ng/mL of NGF for 5, 15, or 30 min. ((A2), (B2), and (C2)) The activity of Akt, JNK, or ERK following 5 min of NGF treatment was abolished by the corresponding specific inhibitor: LY294002 (10 μ M, LY is the abbreviation for LY294002), SP600125 (10 μ M, SP is the abbreviation for SP600125), or PD98059 (10 μ M, PD is the abbreviation for PD98059). ((A3), (A4), (B3), (B4), (C3), and (C4)) The NGF-induced migration of human skin fibroblasts induced by NG was significantly impaired after incubation with LY294002 (10 μ M), SP600125 (10 μ M), or PD98059 (10 μ M). * P

Techniques Used: Migration, Blocking Assay, Activity Assay, Incubation

4) Product Images from "Beta amyloid oligomers and fibrils stimulate differential activation of primary microglia"

Article Title: Beta amyloid oligomers and fibrils stimulate differential activation of primary microglia

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-6-1

Aβ1–42 stimulated a conformation-specific MAP and tyrosine kinase signaling response along with increased COX-2 and CD68 protein levels . Primary mouse microglia were unstimulated (control) or stimulated with 20 μM Aβo or 20 μM Aβf. (A) Cells were lysed after 5 min with RIPA buffer and lysates were separated by 10% SDS-PAGE and Western blotted with anti-phosphotyrosine antibody (4G10), anti-phospho-JNK antibody, anti-JNK antibody (loading control), anti-phospho-ERK antibody, anti-ERK2 antibody (loading control), anti-phospho-p38 antibody, anti-p38 antibody (loading control), anti-phospho-Lyn antibody, or anti-Lyn antibody (loading control). (B) Cells were also lysed after 5 min with RIPA buffer and Syk was immunoprecipitated. Immunoprecipitates were separated by 7% SDS-PAGE and Western blotted with anti-phosphotyrosine antibody (4G10) and anti-Syk antibody. Arrowheads differentiate specific immunoreactivity from IgG heavy chain. (C) Cells were lysed after 24 hrs with RIPA buffer. Cell lysates were separated by 10% SDS-PAGE and Western blotted with anti-COX-2 antibody, anti-CD68 antibody, anti-CD45 antibody and anti-ERK2 antibody (loading control). Antibody binding was visualized by chemiluminescence. Blots are representative of at least three independent experiments.
Figure Legend Snippet: Aβ1–42 stimulated a conformation-specific MAP and tyrosine kinase signaling response along with increased COX-2 and CD68 protein levels . Primary mouse microglia were unstimulated (control) or stimulated with 20 μM Aβo or 20 μM Aβf. (A) Cells were lysed after 5 min with RIPA buffer and lysates were separated by 10% SDS-PAGE and Western blotted with anti-phosphotyrosine antibody (4G10), anti-phospho-JNK antibody, anti-JNK antibody (loading control), anti-phospho-ERK antibody, anti-ERK2 antibody (loading control), anti-phospho-p38 antibody, anti-p38 antibody (loading control), anti-phospho-Lyn antibody, or anti-Lyn antibody (loading control). (B) Cells were also lysed after 5 min with RIPA buffer and Syk was immunoprecipitated. Immunoprecipitates were separated by 7% SDS-PAGE and Western blotted with anti-phosphotyrosine antibody (4G10) and anti-Syk antibody. Arrowheads differentiate specific immunoreactivity from IgG heavy chain. (C) Cells were lysed after 24 hrs with RIPA buffer. Cell lysates were separated by 10% SDS-PAGE and Western blotted with anti-COX-2 antibody, anti-CD68 antibody, anti-CD45 antibody and anti-ERK2 antibody (loading control). Antibody binding was visualized by chemiluminescence. Blots are representative of at least three independent experiments.

Techniques Used: SDS Page, Western Blot, Immunoprecipitation, Binding Assay

5) Product Images from "Intracerebral adult stem cells transplantation increases brain-derived neurotrophic factor levels and protects against phencyclidine-induced social deficit in mice"

Article Title: Intracerebral adult stem cells transplantation increases brain-derived neurotrophic factor levels and protects against phencyclidine-induced social deficit in mice

Journal: Translational Psychiatry

doi: 10.1038/tp.2011.64

Western blot analysis of phosphorylated AKT ( a ) and ERK ( b ) in cortical lysates. Results represent ratio of phosphorylated protein to total protein—each was normalized to actin. The ratio obtained in untreated controls (saline instead of PCP (phencyclidine) and no cell/clozapine treatment) is represented as 100%. * P
Figure Legend Snippet: Western blot analysis of phosphorylated AKT ( a ) and ERK ( b ) in cortical lysates. Results represent ratio of phosphorylated protein to total protein—each was normalized to actin. The ratio obtained in untreated controls (saline instead of PCP (phencyclidine) and no cell/clozapine treatment) is represented as 100%. * P

Techniques Used: Western Blot

6) Product Images from "Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury"

Article Title: Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191034

Effects of Nox4 overexpression mediated by HA vector-based gene transfer on HK-2 cell apoptosis. Virus expressing the HA vector was used as a control. ( A ) Western blot analysis of the effects of HA vector-Nox4 on p38, JNK, and ERK phosphorylation. ( B ) Effects of inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) on cell apoptosis, as measured by caspase 3/7 activity assays. ( C ) Levels of Nox4 protein were measured after inhibition of p38 (SB203580), JNK (SP600125), and ERK (PD98059). The data are the mean ± SD (n = 5). ** p
Figure Legend Snippet: Effects of Nox4 overexpression mediated by HA vector-based gene transfer on HK-2 cell apoptosis. Virus expressing the HA vector was used as a control. ( A ) Western blot analysis of the effects of HA vector-Nox4 on p38, JNK, and ERK phosphorylation. ( B ) Effects of inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) on cell apoptosis, as measured by caspase 3/7 activity assays. ( C ) Levels of Nox4 protein were measured after inhibition of p38 (SB203580), JNK (SP600125), and ERK (PD98059). The data are the mean ± SD (n = 5). ** p

Techniques Used: Over Expression, Plasmid Preparation, Expressing, Western Blot, Activity Assay, Inhibition

Western blot analysis showing intracellular Nox4 signaling in iohexol-induced apoptosis in HK-2 cells. HK-2 cells were incubated with iohexol for the indicated times (0 min, 5 min, 30 min, 1 h, 2 h, or 3 h). For examination of the long-term viability of HK-2 cells after iohexol exposure, the medium containing iohexol was removed after 5 min, 0.5 h, 1 h, 2 h, or 3 h and then replaced with fresh serum-free medium for 24 h, 23.5 h, 23 h, 22 h, or 21 h (5 min/24 h, 0.5/23.5 h, 1/23 h, 2/22 h or 3/21 h). ( A ) Effects of Nox4 on phosphorylation of MAPK family members (p38, JNK, and ERK), bax, bcl-2, and p65. ( B and C ) Effects of Nox4 knockdown and GKT137831 pretreatment on iohexol-induced apoptosis.
Figure Legend Snippet: Western blot analysis showing intracellular Nox4 signaling in iohexol-induced apoptosis in HK-2 cells. HK-2 cells were incubated with iohexol for the indicated times (0 min, 5 min, 30 min, 1 h, 2 h, or 3 h). For examination of the long-term viability of HK-2 cells after iohexol exposure, the medium containing iohexol was removed after 5 min, 0.5 h, 1 h, 2 h, or 3 h and then replaced with fresh serum-free medium for 24 h, 23.5 h, 23 h, 22 h, or 21 h (5 min/24 h, 0.5/23.5 h, 1/23 h, 2/22 h or 3/21 h). ( A ) Effects of Nox4 on phosphorylation of MAPK family members (p38, JNK, and ERK), bax, bcl-2, and p65. ( B and C ) Effects of Nox4 knockdown and GKT137831 pretreatment on iohexol-induced apoptosis.

Techniques Used: Western Blot, Incubation

7) Product Images from "Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1"

Article Title: Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1

Journal: Oncotarget

doi:

Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p
Figure Legend Snippet: Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p

Techniques Used: Migration, Western Blot, Incubation, Positive Control, Activity Assay, Dominant Negative Mutation, Expressing, shRNA, Plasmid Preparation

8) Product Images from "The EGF/hnRNP Q1 axis is involved in tumorigenesis via the regulation of cell cycle-related genes"

Article Title: The EGF/hnRNP Q1 axis is involved in tumorigenesis via the regulation of cell cycle-related genes

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-018-0101-6

The mTOR and ERK pathways regulate the hnRNP Q1-induced translation of Aurora-A mRNA following stimulation with EGF. a SW480 cells were transfected with GFP-hnRNP Q1 for 24 h. Serum-starved cells were pre-treated with rapamycin (100 nM) for 2 h and then were treated with EGF (10 nM) for an additional 2 h. The level of Aurora-A protein was evaluated using western blot analysis and was quantified as a ratio. b Quantification of the expression level of Aurora-A mRNA in GFP-hnRNP Q1-expressing cells treated with rapamycin and EGF, as described in ( a ). c The translational efficiency of the Aurora-A mRNA was determined using S6-IP and was quantitated using RT-qPCR. d , e GFP-hnRNP Q1-expressing SW480 cells were serum-starved and then pre-treated with U0126 (10 μM) for 30 min followed by treatment with EGF for another 2 h. Cell lysates were collected to determine the protein expression of Aurora-A by western blot analysis ( d ) and the mRNA expression of Aurora-A using RT-qPCR ( e ). f , g GFP-hnRNP Q1-expressing cells were treated with U0126 and then EGF, as described above. The cell lysates were then collected for S6-IP ( f ) or RIP assays using anti-GFP antibodies ( g ). The bound Aurora-A mRNA was measured using RT-qPCR. The mean ± S.D. was obtained from three independent experiments. (* p
Figure Legend Snippet: The mTOR and ERK pathways regulate the hnRNP Q1-induced translation of Aurora-A mRNA following stimulation with EGF. a SW480 cells were transfected with GFP-hnRNP Q1 for 24 h. Serum-starved cells were pre-treated with rapamycin (100 nM) for 2 h and then were treated with EGF (10 nM) for an additional 2 h. The level of Aurora-A protein was evaluated using western blot analysis and was quantified as a ratio. b Quantification of the expression level of Aurora-A mRNA in GFP-hnRNP Q1-expressing cells treated with rapamycin and EGF, as described in ( a ). c The translational efficiency of the Aurora-A mRNA was determined using S6-IP and was quantitated using RT-qPCR. d , e GFP-hnRNP Q1-expressing SW480 cells were serum-starved and then pre-treated with U0126 (10 μM) for 30 min followed by treatment with EGF for another 2 h. Cell lysates were collected to determine the protein expression of Aurora-A by western blot analysis ( d ) and the mRNA expression of Aurora-A using RT-qPCR ( e ). f , g GFP-hnRNP Q1-expressing cells were treated with U0126 and then EGF, as described above. The cell lysates were then collected for S6-IP ( f ) or RIP assays using anti-GFP antibodies ( g ). The bound Aurora-A mRNA was measured using RT-qPCR. The mean ± S.D. was obtained from three independent experiments. (* p

Techniques Used: Transfection, Western Blot, Expressing, Quantitative RT-PCR

9) Product Images from "Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase"

Article Title: Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065235

Activation of p38 MAPK by pathogenic SOD1 in axoplasm and mouse spinal cord. (a) Immunoblots with phosphoantibodies show activation of p38 (p-p38) in axoplasms perfused with G93A-SOD1 (G93A), compared to WT-SOD1 (WT). No changes were found in activities of GSK3 (p-GSK3), JNK (p-JNK) or ERK (p-ERK) with perfusion of SOD1. An SOD1 antibody confirmed that similar levels of WT-SOD1 and G93A-SOD1 were perfused, and kinesin-1 (KHC) antibodies serve as a loading control for axoplasmic protein. Representative results from three independent experiments (Squids 1–3) are shown. (b) Quantitation of blots reveals a 3–4 fold increase in p-p38 with G93A SOD1, compared to WT-SOD1 ( n = 8; p≤0.05 (#) by a t-test). No significant differences were found in levels of activated GSK3 ( n = 3) or ERK ( n = 3) (c) Increased activation of p38, but not ERK was also seen in axoplasms perfused with G85R-SOD1 (G85R) polypeptides. (d) Phosphorylation of both neurofilaments (NF) and p38 MAPK was analyzed in spinal cords of age-matched (50 days old) non-transgenic (Naïve), human WT-SOD1 (WT) transgenic and human G93A-SOD1 (G93A) transgenic mice using phosphorylation sensitive antibodies. Kinesin heavy chain (KHC) blots show similar levels of protein loading. SMI32 antibodies recognize a dephosphorylated epitope in NFH that can be phosphorylated by MAPKs, whereas SMI31 recognizes an epitope not affected by phosphorylation with MAPKs [34] . SMI31 immunoreactivity showed similar levels in all mice and serves as a second loading control. In contrast, SMI32 reactivity is reduced in G93A-SOD1 mice, but not naïve and WT-SOD1 mice, suggesting increased phosphorylation of NFs by MAPKs in FALS. Accordingly, p38 activity (p-p38) was increased in spinal cord of G93A-SOD1 mice, with a slight activation in WT-SOD1 mice.
Figure Legend Snippet: Activation of p38 MAPK by pathogenic SOD1 in axoplasm and mouse spinal cord. (a) Immunoblots with phosphoantibodies show activation of p38 (p-p38) in axoplasms perfused with G93A-SOD1 (G93A), compared to WT-SOD1 (WT). No changes were found in activities of GSK3 (p-GSK3), JNK (p-JNK) or ERK (p-ERK) with perfusion of SOD1. An SOD1 antibody confirmed that similar levels of WT-SOD1 and G93A-SOD1 were perfused, and kinesin-1 (KHC) antibodies serve as a loading control for axoplasmic protein. Representative results from three independent experiments (Squids 1–3) are shown. (b) Quantitation of blots reveals a 3–4 fold increase in p-p38 with G93A SOD1, compared to WT-SOD1 ( n = 8; p≤0.05 (#) by a t-test). No significant differences were found in levels of activated GSK3 ( n = 3) or ERK ( n = 3) (c) Increased activation of p38, but not ERK was also seen in axoplasms perfused with G85R-SOD1 (G85R) polypeptides. (d) Phosphorylation of both neurofilaments (NF) and p38 MAPK was analyzed in spinal cords of age-matched (50 days old) non-transgenic (Naïve), human WT-SOD1 (WT) transgenic and human G93A-SOD1 (G93A) transgenic mice using phosphorylation sensitive antibodies. Kinesin heavy chain (KHC) blots show similar levels of protein loading. SMI32 antibodies recognize a dephosphorylated epitope in NFH that can be phosphorylated by MAPKs, whereas SMI31 recognizes an epitope not affected by phosphorylation with MAPKs [34] . SMI31 immunoreactivity showed similar levels in all mice and serves as a second loading control. In contrast, SMI32 reactivity is reduced in G93A-SOD1 mice, but not naïve and WT-SOD1 mice, suggesting increased phosphorylation of NFs by MAPKs in FALS. Accordingly, p38 activity (p-p38) was increased in spinal cord of G93A-SOD1 mice, with a slight activation in WT-SOD1 mice.

Techniques Used: Activation Assay, Western Blot, Quantitation Assay, Transgenic Assay, Mouse Assay, Activity Assay

10) Product Images from "Effects of Oleacein on High-Fat Diet-Dependent Steatosis, Weight Gain, and Insulin Resistance in Mice"

Article Title: Effects of Oleacein on High-Fat Diet-Dependent Steatosis, Weight Gain, and Insulin Resistance in Mice

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2018.00116

Effects of oleacein on insulin levels, liver steatosis, and Western blot analyses of SREBP-1 and ERK. (A) Hematoxylin and eosin staining of liver sections (20× magnification). Liver of obese mice fed with high-fat diet (HFD) was rich in fat vacuoles (indicated by the arrows). For steatosis evaluation, a numerical score was assigned to each section, on the basis of the percentage of vacuolated cells as calculated for each mouse liver: 1 = 1–25%, 2 = 30–65%, 3 = > 70%. (B) Protein expression levels of SREBP-1, ERK, and p-ERK were detected in liver from oleacein-treated and untreated mice fed with HFD and mice fed with NCD. GAPDH, control of protein loading. (C) .” Insulin levels were measured after 13 weeks of feeding with HFD with and without oleacein. Values are expressed as mean ± SD. * p
Figure Legend Snippet: Effects of oleacein on insulin levels, liver steatosis, and Western blot analyses of SREBP-1 and ERK. (A) Hematoxylin and eosin staining of liver sections (20× magnification). Liver of obese mice fed with high-fat diet (HFD) was rich in fat vacuoles (indicated by the arrows). For steatosis evaluation, a numerical score was assigned to each section, on the basis of the percentage of vacuolated cells as calculated for each mouse liver: 1 = 1–25%, 2 = 30–65%, 3 = > 70%. (B) Protein expression levels of SREBP-1, ERK, and p-ERK were detected in liver from oleacein-treated and untreated mice fed with HFD and mice fed with NCD. GAPDH, control of protein loading. (C) .” Insulin levels were measured after 13 weeks of feeding with HFD with and without oleacein. Values are expressed as mean ± SD. * p

Techniques Used: Western Blot, Staining, Mouse Assay, Expressing

11) Product Images from "Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)"

Article Title: Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)

Journal: Journal of Oncology

doi: 10.1155/2009/627840

(a) Western blot of NF-kB : NF-kB expression in control (lane C) and ATRA treated (lane E) MCF-7 cells was observed by western blot analysis as before using anti-NF- κ B primary antibody. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells. (b), (c), and (d) Immunoblot assay of ERK, p-ERK, and VEGF: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (lane C) and presence of 30 μ M ATRA (lane E) for 24 hours in SFCM. Cells were collected and extracted in cell extraction buffer. ERK and PI-3K were immunoprecipitated from 150 μ g of protein from both control and ATRA-treated cell extract with anti-ERK protein G Agarose bead (Figures 7(b) and 7(c) ) and with anti-VEGF protein G Agarose bead ( Figure 7(d) ), keeping the samples for overnight at 4°C with shaking. In each case the resultant immune complex was washed thrice with PBS and the respective protein bound with antibody were eluted from the agarose bead using 1X sample buffer. Samples were then incubated in β -mercaptoethanol for 10 minutes at 90°C. Samples were subjected to electrophoresis on 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane by Western Blot. The membranes were incubated with anti-ERK ( Figure 7(b) ), anti-phospho ERK ( Figure 7(c) ), and anti-VEGF antibody ( Figure 7(d) ), respectively. The immunoblots were then incubated with alkaline phosphatase-coupled secondary antibodies and bands were visualized by NBT/BCIP substrate. Ig-G ( Figure 7(e) ) was used to confirm equal loading. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells.
Figure Legend Snippet: (a) Western blot of NF-kB : NF-kB expression in control (lane C) and ATRA treated (lane E) MCF-7 cells was observed by western blot analysis as before using anti-NF- κ B primary antibody. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells. (b), (c), and (d) Immunoblot assay of ERK, p-ERK, and VEGF: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (lane C) and presence of 30 μ M ATRA (lane E) for 24 hours in SFCM. Cells were collected and extracted in cell extraction buffer. ERK and PI-3K were immunoprecipitated from 150 μ g of protein from both control and ATRA-treated cell extract with anti-ERK protein G Agarose bead (Figures 7(b) and 7(c) ) and with anti-VEGF protein G Agarose bead ( Figure 7(d) ), keeping the samples for overnight at 4°C with shaking. In each case the resultant immune complex was washed thrice with PBS and the respective protein bound with antibody were eluted from the agarose bead using 1X sample buffer. Samples were then incubated in β -mercaptoethanol for 10 minutes at 90°C. Samples were subjected to electrophoresis on 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane by Western Blot. The membranes were incubated with anti-ERK ( Figure 7(b) ), anti-phospho ERK ( Figure 7(c) ), and anti-VEGF antibody ( Figure 7(d) ), respectively. The immunoblots were then incubated with alkaline phosphatase-coupled secondary antibodies and bands were visualized by NBT/BCIP substrate. Ig-G ( Figure 7(e) ) was used to confirm equal loading. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells.

Techniques Used: Western Blot, Expressing, Immunoprecipitation, Incubation, Electrophoresis, SDS Page

12) Product Images from "ERK expression and its correlation with STAT1 in esophageal squamous cell carcinoma"

Article Title: ERK expression and its correlation with STAT1 in esophageal squamous cell carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.16902

p-ERK, ERK and STAT1 expression in normal epithelial, carcinoma in situ (CIS) and ESCC patient samples The esophageal normal epithelial, CIS and carcinoma tissues were stain by Hematoxylin and eosin (H E). By immunohistochemistry applied to formalin-fixed paraffin-embedded tissues, variable levels of p-ERK, ERK and STAT1 were detected in normal esophageal, CIS and carcinoma examined. (IHC stain, scale bar, 50 μM).
Figure Legend Snippet: p-ERK, ERK and STAT1 expression in normal epithelial, carcinoma in situ (CIS) and ESCC patient samples The esophageal normal epithelial, CIS and carcinoma tissues were stain by Hematoxylin and eosin (H E). By immunohistochemistry applied to formalin-fixed paraffin-embedded tissues, variable levels of p-ERK, ERK and STAT1 were detected in normal esophageal, CIS and carcinoma examined. (IHC stain, scale bar, 50 μM).

Techniques Used: Expressing, In Situ, Staining, Immunohistochemistry, Formalin-fixed Paraffin-Embedded

ERK mediated STAT1 expression in ESCC ( A ) KYSE510 and KYSE150 cell lines were treated with increasing doses of U0126 for 90 min. Western blot analysis of p-STAT1, STAT1, p-ERK and ERK in total cell lysates are shown. ( B ) KYSE150 cells were transfected with a constitutive-active MEK expression (HA-ca-MEK) plasmid, and endogenous protein levels of p-STAT1 and STAT1 in the lysates were determined by immunoblotting. Similar results were observed in three independent experiments.
Figure Legend Snippet: ERK mediated STAT1 expression in ESCC ( A ) KYSE510 and KYSE150 cell lines were treated with increasing doses of U0126 for 90 min. Western blot analysis of p-STAT1, STAT1, p-ERK and ERK in total cell lysates are shown. ( B ) KYSE150 cells were transfected with a constitutive-active MEK expression (HA-ca-MEK) plasmid, and endogenous protein levels of p-STAT1 and STAT1 in the lysates were determined by immunoblotting. Similar results were observed in three independent experiments.

Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation

Kaplan-Meier curves of esophageal cancer patients in different p-ERK, ERK and STAT1 sub-groups ( A ) By Kaplan-Meier analysis, the significant correlation between overall survival and the expression level of p-ERK was found, when the two groups were defined as p-ERK high and p-ERK low . Similar result was obtained in ERK high/low expression patient groups ( B ). The cohort patients were divided into four sub groups by the STAT1 and p-ERK expression level, we found that patients with p-ERK low /STAT1 high survived significantly longer than other subgroups ( C ) and those with p-ERK high /STAT1 low survived significantly shorter than others ( D ). By Kaplan-Meier analysis, significant correlation between overall survivals was found when the cohort patients were divided into four sub groups by the STAT1 and p-ERK/ERK expression level ( E , F ).
Figure Legend Snippet: Kaplan-Meier curves of esophageal cancer patients in different p-ERK, ERK and STAT1 sub-groups ( A ) By Kaplan-Meier analysis, the significant correlation between overall survival and the expression level of p-ERK was found, when the two groups were defined as p-ERK high and p-ERK low . Similar result was obtained in ERK high/low expression patient groups ( B ). The cohort patients were divided into four sub groups by the STAT1 and p-ERK expression level, we found that patients with p-ERK low /STAT1 high survived significantly longer than other subgroups ( C ) and those with p-ERK high /STAT1 low survived significantly shorter than others ( D ). By Kaplan-Meier analysis, significant correlation between overall survivals was found when the cohort patients were divided into four sub groups by the STAT1 and p-ERK/ERK expression level ( E , F ).

Techniques Used: Expressing

The IHC score of p-ERK ( A ), ERK ( B ) and STAT1 ( C ) in normal tissues, CIS and ESCC are shown. (* p
Figure Legend Snippet: The IHC score of p-ERK ( A ), ERK ( B ) and STAT1 ( C ) in normal tissues, CIS and ESCC are shown. (* p

Techniques Used: Immunohistochemistry

13) Product Images from "Inhibition of odontogenic differentiation of human dental pulp cells by dental resin monomers"

Article Title: Inhibition of odontogenic differentiation of human dental pulp cells by dental resin monomers

Journal: Biomaterials Research

doi: 10.1186/s40824-015-0030-6

Effects of TEGDMA, HEMA, and H 2 O 2 on the expression of MAP kinase proteins (ERK, JNK, and p38). Cells were treated with 0.5 mM TEGDMA, 2 mM HEMA, and 0.2 mM H 2 O 2 for 1, 3, and 6 hrs. A representative image from three independent experiments is shown.
Figure Legend Snippet: Effects of TEGDMA, HEMA, and H 2 O 2 on the expression of MAP kinase proteins (ERK, JNK, and p38). Cells were treated with 0.5 mM TEGDMA, 2 mM HEMA, and 0.2 mM H 2 O 2 for 1, 3, and 6 hrs. A representative image from three independent experiments is shown.

Techniques Used: Expressing

14) Product Images from "The Arf GAP AGAP2 interacts with β-arrestin2 and regulates β2-adrenergic receptor recycling and ERK activation"

Article Title: The Arf GAP AGAP2 interacts with β-arrestin2 and regulates β2-adrenergic receptor recycling and ERK activation

Journal: The Biochemical journal

doi: 10.1042/BJ20121004

AGAP2 enhanced β 2 AR-induced ERK activation ( A ) AGAP2 overexpression enhanced β 2 AR-induced ERK activation. HEK-293 cells stably overexpressing empty vector or FLAG–AGAP2 were starved in Opti-MEM overnight and treated with ISO (10 μ M) for the indicated times. Cell lysates were centrifuged and used for detection of phospho-ERK by Western blotting. Total ERK and FLAG–AGAP2 were also detected by Western blotting. ( B ) Quantification of phospho-ERK signals at different times by densitometry. * P
Figure Legend Snippet: AGAP2 enhanced β 2 AR-induced ERK activation ( A ) AGAP2 overexpression enhanced β 2 AR-induced ERK activation. HEK-293 cells stably overexpressing empty vector or FLAG–AGAP2 were starved in Opti-MEM overnight and treated with ISO (10 μ M) for the indicated times. Cell lysates were centrifuged and used for detection of phospho-ERK by Western blotting. Total ERK and FLAG–AGAP2 were also detected by Western blotting. ( B ) Quantification of phospho-ERK signals at different times by densitometry. * P

Techniques Used: Activation Assay, Over Expression, Stable Transfection, Plasmid Preparation, Western Blot

15) Product Images from "Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of ZC3H12A expression"

Article Title: Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of ZC3H12A expression

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-11-14

Regulation of expression of human MCPIP . A , control MOCK cells and B , mIκB cells with disrupted activation of NFκB were treated with IL-1β (15 ng/ml) and PMA (100 nM). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 5-8 (A and B)). RNA was isolated after 2 h and subjected to northern blot analysis using MCPIP cDNA (top panel) or GAPDH cDNA (middle panel) as probes. Bottom panel - activation of ERK in MOCK and mIkB cells by IL-1β or its inhibition by U0126 was checked by western blot analysis. Cells were stimulated by IL-1β (15 ng/ml) for 30 min (line 2 and 4). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 3-4). Line 1 - lysate from control cells. C , human macrophages were treated with IL-1β (15 ng/ml) with or without U0126 (10 μM, 30 min prior IL-1β stimulation). Changes in ZC3H12A expression were measured by Real Time PCR. The results are means ± SD of three independent experiments.
Figure Legend Snippet: Regulation of expression of human MCPIP . A , control MOCK cells and B , mIκB cells with disrupted activation of NFκB were treated with IL-1β (15 ng/ml) and PMA (100 nM). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 5-8 (A and B)). RNA was isolated after 2 h and subjected to northern blot analysis using MCPIP cDNA (top panel) or GAPDH cDNA (middle panel) as probes. Bottom panel - activation of ERK in MOCK and mIkB cells by IL-1β or its inhibition by U0126 was checked by western blot analysis. Cells were stimulated by IL-1β (15 ng/ml) for 30 min (line 2 and 4). To some experimental groups U0126 (10 μM) was added 30 min prior IL-1β stimulation (lanes 3-4). Line 1 - lysate from control cells. C , human macrophages were treated with IL-1β (15 ng/ml) with or without U0126 (10 μM, 30 min prior IL-1β stimulation). Changes in ZC3H12A expression were measured by Real Time PCR. The results are means ± SD of three independent experiments.

Techniques Used: Expressing, Activation Assay, Isolation, Northern Blot, Inhibition, Western Blot, Real-time Polymerase Chain Reaction

16) Product Images from "Induction of Thyroid Gene Expression and Radioiodine Uptake in Thyroid Cancer Cells by Targeting Major Signaling Pathways"

Article Title: Induction of Thyroid Gene Expression and Radioiodine Uptake in Thyroid Cancer Cells by Targeting Major Signaling Pathways

Journal: The Journal of Clinical Endocrinology and Metabolism

doi: 10.1210/jc.2009-1888

Effects of various inhibitors on the MAPK pathway, PI3K/Akt/mTOR pathway, and HDAC in thyroid cancer cells. Inhibition of ERK and p70S6K phosphorylation and histone deacetylation was achieved using the MEK-specific inhibitor RDEA119, mTOR-specific inhibitor
Figure Legend Snippet: Effects of various inhibitors on the MAPK pathway, PI3K/Akt/mTOR pathway, and HDAC in thyroid cancer cells. Inhibition of ERK and p70S6K phosphorylation and histone deacetylation was achieved using the MEK-specific inhibitor RDEA119, mTOR-specific inhibitor

Techniques Used: Inhibition

17) Product Images from "Epstein-Barr Virus LMP1 Modulates Lipid Raft Microdomains and the Vimentin Cytoskeleton for Signal Transduction and Transformation"

Article Title: Epstein-Barr Virus LMP1 Modulates Lipid Raft Microdomains and the Vimentin Cytoskeleton for Signal Transduction and Transformation

Journal: Journal of Virology

doi: 10.1128/JVI.02519-12

LMP1 modulation of membrane rafts and the vimentin cytoskeleton to activate Akt and ERK signal transduction pathways. LMP1 self-aggregates in lipid raft microdomains rich in cholesterol and sphingolipids. LMP1 expression leads to the recruitment of PI3K
Figure Legend Snippet: LMP1 modulation of membrane rafts and the vimentin cytoskeleton to activate Akt and ERK signal transduction pathways. LMP1 self-aggregates in lipid raft microdomains rich in cholesterol and sphingolipids. LMP1 expression leads to the recruitment of PI3K

Techniques Used: Transduction, Expressing

Vimentin is important for the ability of LMP1 to transform cells and activate ERK and Akt. Rat-1 cells stably expressing an shRNA against vimentin (shVIM) or scrambled control (pGIVZ) were transduced with retrovirus particles expressing HA-LMP1 (A) or
Figure Legend Snippet: Vimentin is important for the ability of LMP1 to transform cells and activate ERK and Akt. Rat-1 cells stably expressing an shRNA against vimentin (shVIM) or scrambled control (pGIVZ) were transduced with retrovirus particles expressing HA-LMP1 (A) or

Techniques Used: Stable Transfection, Expressing, shRNA, Transduction

Vimentin organization is essential for LMP1-mediated ERK activation. (A) pBabe or LMP1 stable Rat-1 cells were serum starved for 1 h, treated for 1 h with the indicated compounds, and then analyzed by immunoblot for the activation of ERK and Akt with
Figure Legend Snippet: Vimentin organization is essential for LMP1-mediated ERK activation. (A) pBabe or LMP1 stable Rat-1 cells were serum starved for 1 h, treated for 1 h with the indicated compounds, and then analyzed by immunoblot for the activation of ERK and Akt with

Techniques Used: Activation Assay

Lipid raft disruption inhibits LMP1-mediated Akt and ERK activation. C666 (A) or Rat-1 (B) cells expressing pBabe or LMP1 were serum starved for 1 h and then treated with vehicle (NT) or MβCD for the indicated times. After treatment, cell lysates
Figure Legend Snippet: Lipid raft disruption inhibits LMP1-mediated Akt and ERK activation. C666 (A) or Rat-1 (B) cells expressing pBabe or LMP1 were serum starved for 1 h and then treated with vehicle (NT) or MβCD for the indicated times. After treatment, cell lysates

Techniques Used: Activation Assay, Expressing

18) Product Images from "A Natural Dietary Supplement with a Combination of Nutrients Prevents Neurodegeneration Induced by a High Fat Diet in Mice"

Article Title: A Natural Dietary Supplement with a Combination of Nutrients Prevents Neurodegeneration Induced by a High Fat Diet in Mice

Journal: Nutrients

doi: 10.3390/nu10091130

Nitric and oxidative stress and lipid peroxidation in the brains of HFD-mice were prevented by NDS treatment. ( A ) Nitrite concentration in STD, untreated-HFD and treated-HFD brains; ( B ) Levels of ROS in STD, untreated-HFD and treated-HFD brains ( C ) Lipid peroxidation levels in STD, untreated-HFD and treated-HFD brains; ( D ) Western blot of proteins extracted from brains of STD, untreated-HFD and treated-HFD mice and incubated with anti-p-ERK, anti-H-Oxy, anti-i-NOS, anti-HSP60 and anti-β-Actin (loading control); ( E ) Quantification of immunoreactivity was performed using densitometric analysis; ( F ) Immunofluorescence of superficial and deep cerebral cortex sections of STD, untreated-HFD and treated-HFD mice incubated with anti-phospho-ERK; ( G ) Schematic representation of superficial (i) and deep (ii) cerebral cortex positive areas; ( H ) Brain map indicating the levels of positive p-ERK staining. Representative images from 3 animals per group are shown. Bar 20 μm. Data are the mean values ± SD ( n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.
Figure Legend Snippet: Nitric and oxidative stress and lipid peroxidation in the brains of HFD-mice were prevented by NDS treatment. ( A ) Nitrite concentration in STD, untreated-HFD and treated-HFD brains; ( B ) Levels of ROS in STD, untreated-HFD and treated-HFD brains ( C ) Lipid peroxidation levels in STD, untreated-HFD and treated-HFD brains; ( D ) Western blot of proteins extracted from brains of STD, untreated-HFD and treated-HFD mice and incubated with anti-p-ERK, anti-H-Oxy, anti-i-NOS, anti-HSP60 and anti-β-Actin (loading control); ( E ) Quantification of immunoreactivity was performed using densitometric analysis; ( F ) Immunofluorescence of superficial and deep cerebral cortex sections of STD, untreated-HFD and treated-HFD mice incubated with anti-phospho-ERK; ( G ) Schematic representation of superficial (i) and deep (ii) cerebral cortex positive areas; ( H ) Brain map indicating the levels of positive p-ERK staining. Representative images from 3 animals per group are shown. Bar 20 μm. Data are the mean values ± SD ( n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.

Techniques Used: Mouse Assay, Concentration Assay, Western Blot, Incubation, Immunofluorescence, Staining

19) Product Images from "AREG mediates the epithelial-mesenchymal transition in pancreatic cancer cells via the EGFR/ERK/NF-κB signalling pathway"

Article Title: AREG mediates the epithelial-mesenchymal transition in pancreatic cancer cells via the EGFR/ERK/NF-κB signalling pathway

Journal: Oncology Reports

doi: 10.3892/or.2020.7523

AREG activates the EGFR/ERK signalling pathway in pancreatic cancer cells. (A) Phosphorylation and total protein expression levels of EGFR, STAT3, AKT, and ERK in AsPC-1 cells transfected with AREG siRNA (siAR-390) or control siRNA (NC). (B) In PANC-1 cells, the exogenous treatment of AREG induced EGFR signalling as revealed by the varying degrees of activation of the p-ERK, p-AKT and p-STAT3 levels. The enhancement effect was blocked by PD153035 (an inhibitor of EGF receptor tyrosine kinase, 5 µM) or U0126 (an inhibitor of MEK1/2, 20 µM). (C) Activation of ERK in response to AREG in PANC-1 cells. AREG stimulation resulted in a significant induction of p-ERK, which stabilized at 30 min. Graphs are presented as the relative density of the phosphoprotein vs. the total protein. *P
Figure Legend Snippet: AREG activates the EGFR/ERK signalling pathway in pancreatic cancer cells. (A) Phosphorylation and total protein expression levels of EGFR, STAT3, AKT, and ERK in AsPC-1 cells transfected with AREG siRNA (siAR-390) or control siRNA (NC). (B) In PANC-1 cells, the exogenous treatment of AREG induced EGFR signalling as revealed by the varying degrees of activation of the p-ERK, p-AKT and p-STAT3 levels. The enhancement effect was blocked by PD153035 (an inhibitor of EGF receptor tyrosine kinase, 5 µM) or U0126 (an inhibitor of MEK1/2, 20 µM). (C) Activation of ERK in response to AREG in PANC-1 cells. AREG stimulation resulted in a significant induction of p-ERK, which stabilized at 30 min. Graphs are presented as the relative density of the phosphoprotein vs. the total protein. *P

Techniques Used: Expressing, Transfection, Activation Assay

20) Product Images from "Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells"

Article Title: Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

Effect of inhibitors of MAPK pathways on NECA-dependent IL-13 and VEGF secretion from BMMCs and HMC-1 cells. A , Western blot analysis of phosphorylation of ERK and p38 MAPK (phospho-ERK and phospho-p38, respectively) in BMMCs and HMC-1 cells that were either not stimulated (−) or stimulated with 10 μ M NECA for the indicated time period. Equal loading of proteins was verified by reprobing the same blot with total ERK and p38 MAPK Abs (ERK and p38, respectively). Blots are representative of three experiments. Effects of MEK inhibitor UO126 (●) and its inactive analog UO124 (○) on IL-13 ( B ) or VEGF ( D ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. Effects of p38 inhibitor SB202190 (●) and its inactive analog SB202474 (○) on IL-13 ( C ) or VEGF ( E ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. F , Effects of MEK inhibitor UO126 (10 μ M), p38 inhibitor SB202190 (10 μ M), and their inactive analogs UO124 (10 μ M) and SB202474 (10 μ M), respectively, on IL-13 secretion from BMMCs stimulated with 10 μ M NECA. Significant difference ( p
Figure Legend Snippet: Effect of inhibitors of MAPK pathways on NECA-dependent IL-13 and VEGF secretion from BMMCs and HMC-1 cells. A , Western blot analysis of phosphorylation of ERK and p38 MAPK (phospho-ERK and phospho-p38, respectively) in BMMCs and HMC-1 cells that were either not stimulated (−) or stimulated with 10 μ M NECA for the indicated time period. Equal loading of proteins was verified by reprobing the same blot with total ERK and p38 MAPK Abs (ERK and p38, respectively). Blots are representative of three experiments. Effects of MEK inhibitor UO126 (●) and its inactive analog UO124 (○) on IL-13 ( B ) or VEGF ( D ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. Effects of p38 inhibitor SB202190 (●) and its inactive analog SB202474 (○) on IL-13 ( C ) or VEGF ( E ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. F , Effects of MEK inhibitor UO126 (10 μ M), p38 inhibitor SB202190 (10 μ M), and their inactive analogs UO124 (10 μ M) and SB202474 (10 μ M), respectively, on IL-13 secretion from BMMCs stimulated with 10 μ M NECA. Significant difference ( p

Techniques Used: Western Blot

21) Product Images from "Regulatory T cells trigger effector T cell DNA damage and senescence caused by metabolic competition"

Article Title: Regulatory T cells trigger effector T cell DNA damage and senescence caused by metabolic competition

Journal: Nature Communications

doi: 10.1038/s41467-017-02689-5

MAPK cooperates with STAT to control T-cell senescence mediated by Treg cells. a Pretreatment of naive CD4 + T cells with an ATM inhibitor KU55933 significantly prevented P38 and ERK phosphorylation in responder T cells treated by nTreg cells using western blot analyses. Anti-CD3 activated CD4 + T cells were pretreated with or without KU55933 (10 μM) for 1 day, and then co-cultured with nTreg cells for different time points. Protein levels of phosphorylated P38 and ERK shown in the right histogram were quantitatively analyzed and compared against the GAPDH expression levels with a densitometer. Results shown in the histogram are mean ± SD from three independent experiments. ** p
Figure Legend Snippet: MAPK cooperates with STAT to control T-cell senescence mediated by Treg cells. a Pretreatment of naive CD4 + T cells with an ATM inhibitor KU55933 significantly prevented P38 and ERK phosphorylation in responder T cells treated by nTreg cells using western blot analyses. Anti-CD3 activated CD4 + T cells were pretreated with or without KU55933 (10 μM) for 1 day, and then co-cultured with nTreg cells for different time points. Protein levels of phosphorylated P38 and ERK shown in the right histogram were quantitatively analyzed and compared against the GAPDH expression levels with a densitometer. Results shown in the histogram are mean ± SD from three independent experiments. ** p

Techniques Used: Western Blot, Cell Culture, Expressing

22) Product Images from "Effect and Mechanism of Total Flavonoids Extracted from Cotinus coggygria against Glioblastoma Cancer In Vitro and In Vivo"

Article Title: Effect and Mechanism of Total Flavonoids Extracted from Cotinus coggygria against Glioblastoma Cancer In Vitro and In Vivo

Journal: BioMed Research International

doi: 10.1155/2015/856349

Apoptosis induced by CCF was associated with the PI3K/AKT/ERK signaling pathway. (a) Phosphorylation of Akt (Ser473) was significantly lower in U87, U251, and DBTRG-05MG glioblastoma cells treated with CCF. CCF (100 μ g/mL) induced the downregulation of Akt levels in U87, U251, and DBTRG-05MG glioblastoma cells. (b) Compared with the control, CCF had no significant effects on ERK levels, and phospho-ERK levels were downregulated by CCF in U87, U251, and DBTRG-05MG glioblastoma cells. ∗ P
Figure Legend Snippet: Apoptosis induced by CCF was associated with the PI3K/AKT/ERK signaling pathway. (a) Phosphorylation of Akt (Ser473) was significantly lower in U87, U251, and DBTRG-05MG glioblastoma cells treated with CCF. CCF (100 μ g/mL) induced the downregulation of Akt levels in U87, U251, and DBTRG-05MG glioblastoma cells. (b) Compared with the control, CCF had no significant effects on ERK levels, and phospho-ERK levels were downregulated by CCF in U87, U251, and DBTRG-05MG glioblastoma cells. ∗ P

Techniques Used:

23) Product Images from "Absence of RIPK3 predicts necroptosis resistance in malignant melanoma"

Article Title: Absence of RIPK3 predicts necroptosis resistance in malignant melanoma

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.240

CD95L/zVAD/IAP antagonist-mediated necroptosis induction and MLKL phosphorylation depends on RIPK1 and RIPK3. ( a ) MLKL phosphorylation in RIPK3 expressing cells depends on RIPK1 and RIPK3. RIPK3-overexpressing A375 melanomas were either pre-treated with IAP antagonist (100 nM), zVAD-fmk (10 μ M), Nec-1 (50 μ M), NSA (10 μ M), RIPK3 kinase inhibitor (GSK'840 and GSK'872) or RIPK1 inhibitors (7-Cl-O-Nec-1 and GSK'481; 10 μ M each) alone or in respective indicated combinations for 1 h followed by CD95L (0.5 U/ml) costimulation for 6 h. Phosphorylation of MLKL or ERK as well as expression of MLKL, RIPK3, RIPK1, ERK, and actin was analysed by western blot analysis as previously described. One of three representative independently performed experiments is shown. ( b ) RIPK3 inhibitors fully protect RIPK3 reconstituted melanomas against CD95L/IAP antagonist-mediated necroptosis. Vector control and RIPK3-expressing cells were either pretreated with IAP antagonist (100 nM), zVAD-fmk (10 μ M), RIPK3 kinase inhibitor (GSK'840 and GSK'872), or RIPK1 inhibitors (7-Cl-O-Nec-1 and GSK'481) alone or in respective combinations followed by CD95L (0.5 U/ml) costimulation for 18–24 h followed by analysis with crystal violet assay as described previously. Summary of multiple independently performed experiments (three experiments) is shown including the S.E.M. of the whole set of experiments
Figure Legend Snippet: CD95L/zVAD/IAP antagonist-mediated necroptosis induction and MLKL phosphorylation depends on RIPK1 and RIPK3. ( a ) MLKL phosphorylation in RIPK3 expressing cells depends on RIPK1 and RIPK3. RIPK3-overexpressing A375 melanomas were either pre-treated with IAP antagonist (100 nM), zVAD-fmk (10 μ M), Nec-1 (50 μ M), NSA (10 μ M), RIPK3 kinase inhibitor (GSK'840 and GSK'872) or RIPK1 inhibitors (7-Cl-O-Nec-1 and GSK'481; 10 μ M each) alone or in respective indicated combinations for 1 h followed by CD95L (0.5 U/ml) costimulation for 6 h. Phosphorylation of MLKL or ERK as well as expression of MLKL, RIPK3, RIPK1, ERK, and actin was analysed by western blot analysis as previously described. One of three representative independently performed experiments is shown. ( b ) RIPK3 inhibitors fully protect RIPK3 reconstituted melanomas against CD95L/IAP antagonist-mediated necroptosis. Vector control and RIPK3-expressing cells were either pretreated with IAP antagonist (100 nM), zVAD-fmk (10 μ M), RIPK3 kinase inhibitor (GSK'840 and GSK'872), or RIPK1 inhibitors (7-Cl-O-Nec-1 and GSK'481) alone or in respective combinations followed by CD95L (0.5 U/ml) costimulation for 18–24 h followed by analysis with crystal violet assay as described previously. Summary of multiple independently performed experiments (three experiments) is shown including the S.E.M. of the whole set of experiments

Techniques Used: Expressing, Western Blot, Plasmid Preparation, Crystal Violet Assay

Dabrafenib, but not Vemurafenib, suppresses DL/IAP antagonist-mediated necroptosis by inhibition of MLKL phosphorylation. ( a and b ) Dabrafenib blocks necroptosis in RIPK3-reconstituted melanomas. Vector control and RIPK3-expressing A375 ( a ) or IGR ( b ) melanomas were either pretreated for 2 h with IAP antagonist (100 nM), zVAD-fmk (10 μ M), Dabrafenib (10 μ M), or Vemurafenib (A375 with 30 μ M and IGR with 10 μ M) alone or in respective combinations followed by CD95L (0.5 U/ml) costimulation for 18–24 h and analysis with crystal violet assay as described previously. Summary of three independently performed experiments is shown. The S.E.M. of the whole set of experiments is depicted. ( c and d ) Dabrafenib but not Vemurafenib inhibits MLKL phosphorylation in RIPK3-reconstituted A375 ( c ) and IGR ( d ) melanomas. RIPK3-expressing A375 ( c ) or IGR ( d ) melanomas were either pretreated for 2 h with IAP antagonist (100 nM), zVAD-fmk (10 μ M), Dabrafenib (10 μ M), or Vemurafenib (A375 with 30 μ M and IGR with 10 μ M) alone or in respective combinations followed by CD95L (0.5 U/ml) costimulation for 6 h. Phosphorylation of MLKL or ERK as well as expression of MLKL, RIPK3, RIPK1, ERK, and actin was analysed by western blot analysis as previously described. One of three independently performed experiments is shown as representation of the results
Figure Legend Snippet: Dabrafenib, but not Vemurafenib, suppresses DL/IAP antagonist-mediated necroptosis by inhibition of MLKL phosphorylation. ( a and b ) Dabrafenib blocks necroptosis in RIPK3-reconstituted melanomas. Vector control and RIPK3-expressing A375 ( a ) or IGR ( b ) melanomas were either pretreated for 2 h with IAP antagonist (100 nM), zVAD-fmk (10 μ M), Dabrafenib (10 μ M), or Vemurafenib (A375 with 30 μ M and IGR with 10 μ M) alone or in respective combinations followed by CD95L (0.5 U/ml) costimulation for 18–24 h and analysis with crystal violet assay as described previously. Summary of three independently performed experiments is shown. The S.E.M. of the whole set of experiments is depicted. ( c and d ) Dabrafenib but not Vemurafenib inhibits MLKL phosphorylation in RIPK3-reconstituted A375 ( c ) and IGR ( d ) melanomas. RIPK3-expressing A375 ( c ) or IGR ( d ) melanomas were either pretreated for 2 h with IAP antagonist (100 nM), zVAD-fmk (10 μ M), Dabrafenib (10 μ M), or Vemurafenib (A375 with 30 μ M and IGR with 10 μ M) alone or in respective combinations followed by CD95L (0.5 U/ml) costimulation for 6 h. Phosphorylation of MLKL or ERK as well as expression of MLKL, RIPK3, RIPK1, ERK, and actin was analysed by western blot analysis as previously described. One of three independently performed experiments is shown as representation of the results

Techniques Used: Inhibition, Plasmid Preparation, Expressing, Crystal Violet Assay, Western Blot

24) Product Images from "Cardioprotective Effects of Telmisartan against Heart Failure in Rats Induced By Experimental Autoimmune Myocarditis through the Modulation of Angiotensin-Converting Enzyme-2/Angiotensin 1-7/Mas Receptor Axis"

Article Title: Cardioprotective Effects of Telmisartan against Heart Failure in Rats Induced By Experimental Autoimmune Myocarditis through the Modulation of Angiotensin-Converting Enzyme-2/Angiotensin 1-7/Mas Receptor Axis

Journal: International Journal of Biological Sciences

doi:

Myocardial expressions of phospho-p38 MAPK, phospho-JNK, p38 MAPK, JNK, phospho-ERK, ERK, phospho-p38MAPKAPK-2 and p38-MAPKAPK-2. [8A] Representative western blots showing specific bands for phospho-p38 MAPK, phospho-JNK, p38 MAPK, JNK, phospho-ERK, ERK, phospho-p38MAPKAPK-2 and p38-MAPKAPK-2 as an internal control. An equal amount of protein sample (30 μg) obtained from whole ventricular homogenate was applied in each lane. These bands are representative of eight separate experiments. [8B-8E], Bar graph showing the densitometric analysis of the above Western blots. The mean density value of phospho-p38 MAPK, phospho-JNK, phospho-ERK and phospho-p38MAPKAPK-2 was expressed as a ratio relative to that of p38 MAPK, JNK, ERK and p38-MAPKAPK-2. Each bar represents the mean ± S.E.M of 4 to 6 rats. Group N, age-matched untreated rats; group EAM, EAM rats administered with vehicle; group Tel-10, EAM rats treated with telmisartan (10 mg/kg/day) respectively; * P
Figure Legend Snippet: Myocardial expressions of phospho-p38 MAPK, phospho-JNK, p38 MAPK, JNK, phospho-ERK, ERK, phospho-p38MAPKAPK-2 and p38-MAPKAPK-2. [8A] Representative western blots showing specific bands for phospho-p38 MAPK, phospho-JNK, p38 MAPK, JNK, phospho-ERK, ERK, phospho-p38MAPKAPK-2 and p38-MAPKAPK-2 as an internal control. An equal amount of protein sample (30 μg) obtained from whole ventricular homogenate was applied in each lane. These bands are representative of eight separate experiments. [8B-8E], Bar graph showing the densitometric analysis of the above Western blots. The mean density value of phospho-p38 MAPK, phospho-JNK, phospho-ERK and phospho-p38MAPKAPK-2 was expressed as a ratio relative to that of p38 MAPK, JNK, ERK and p38-MAPKAPK-2. Each bar represents the mean ± S.E.M of 4 to 6 rats. Group N, age-matched untreated rats; group EAM, EAM rats administered with vehicle; group Tel-10, EAM rats treated with telmisartan (10 mg/kg/day) respectively; * P

Techniques Used: Western Blot

25) Product Images from "The Hexane Fraction of Bursera microphylla A Gray Induces p21-Mediated Antiproliferative and Proapoptotic Effects in Human Cancer–Derived Cell Lines"

Article Title: The Hexane Fraction of Bursera microphylla A Gray Induces p21-Mediated Antiproliferative and Proapoptotic Effects in Human Cancer–Derived Cell Lines

Journal: Integrative Cancer Therapies

doi: 10.1177/1534735416688413

Time course effects of hexane fraction of resin methanol extract (BM-H) on MAPK pathway. Western blot bands represent phosphorylated ERK (p-ERK), total ERK (ERK), actin (as a housekeeping gene for ERK), phosphorylated p38 (p-p38), total p38 (p38), phosphorylated Akt (p-Akt), total Akt (Akt), and the housekeeping gene tubulin (to control both p-38 and Akt). Proteins were extracted from OCI cells treated with the vehicle (control) or with BM-H (BM-H) after 4, 8, 14, or 24 hours. Western blots are representative of 3 independent experiments.
Figure Legend Snippet: Time course effects of hexane fraction of resin methanol extract (BM-H) on MAPK pathway. Western blot bands represent phosphorylated ERK (p-ERK), total ERK (ERK), actin (as a housekeeping gene for ERK), phosphorylated p38 (p-p38), total p38 (p38), phosphorylated Akt (p-Akt), total Akt (Akt), and the housekeeping gene tubulin (to control both p-38 and Akt). Proteins were extracted from OCI cells treated with the vehicle (control) or with BM-H (BM-H) after 4, 8, 14, or 24 hours. Western blots are representative of 3 independent experiments.

Techniques Used: Western Blot

26) Product Images from "Two Closely Related Human Members of Chitinase-like Family, CHI3L1 and CHI3L2, Activate ERK1/2 in 293 and U373 Cells but Have the Different Influence on Cell Proliferation"

Article Title: Two Closely Related Human Members of Chitinase-like Family, CHI3L1 and CHI3L2, Activate ERK1/2 in 293 and U373 Cells but Have the Different Influence on Cell Proliferation

Journal: International Journal of Biological Sciences

doi:

Dose-dependent phosphorylation of ERK1/2 induced by CHI3L1 and CHI3L2. (A) Western-blot analysis of U373 cells with anti-phospho-ERK and pan-anti-ERK antibodies after CHI3L1 or CHI3L2 cell treatment (60 min). (B) Western-blot analysis of U373 cell, 293 cell, and MG-63 cell media or purified CHI3L1 and CHI3L2 proteins with anti-CHI3L1, -CHI3L2 or -ACTB antibodies.
Figure Legend Snippet: Dose-dependent phosphorylation of ERK1/2 induced by CHI3L1 and CHI3L2. (A) Western-blot analysis of U373 cells with anti-phospho-ERK and pan-anti-ERK antibodies after CHI3L1 or CHI3L2 cell treatment (60 min). (B) Western-blot analysis of U373 cell, 293 cell, and MG-63 cell media or purified CHI3L1 and CHI3L2 proteins with anti-CHI3L1, -CHI3L2 or -ACTB antibodies.

Techniques Used: Western Blot, Purification

27) Product Images from "Mitochondrial Ca2+ uptake is essential for synaptic plasticity in pain"

Article Title: Mitochondrial Ca2+ uptake is essential for synaptic plasticity in pain

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.3093-11.2011

A diagrammatic representation of the proposed hypothesis that mitochondrial Ca 2+ uptake is an essential downstream event of [Ca 2+ ] C influx for synaptic plasticity in the spinal cord. During NMDA receptor activation, there is a massive Ca 2+ influx into spinal dorsal horn neurons. Most of the Ca 2+ is rapidly sequestrated by mitochondria. This consequently increases ROS production which leads to activation of intracellular signaling cascades (i.g., PKC, PKA and ERK), which in turn results in synaptic plasticity of the dorsal horn neurons. Glu, glutamate; NMDAR, NMDA receptor; AMPAR, AMPA receptor; [Ca 2+ ] c , cytosolic free Ca 2+ ; [Ca 2+ ] m , mitochondrial Ca 2+ ; ETC, electron transport complex; ROS, reactive oxygen species; circle P, phosphorylation.
Figure Legend Snippet: A diagrammatic representation of the proposed hypothesis that mitochondrial Ca 2+ uptake is an essential downstream event of [Ca 2+ ] C influx for synaptic plasticity in the spinal cord. During NMDA receptor activation, there is a massive Ca 2+ influx into spinal dorsal horn neurons. Most of the Ca 2+ is rapidly sequestrated by mitochondria. This consequently increases ROS production which leads to activation of intracellular signaling cascades (i.g., PKC, PKA and ERK), which in turn results in synaptic plasticity of the dorsal horn neurons. Glu, glutamate; NMDAR, NMDA receptor; AMPAR, AMPA receptor; [Ca 2+ ] c , cytosolic free Ca 2+ ; [Ca 2+ ] m , mitochondrial Ca 2+ ; ETC, electron transport complex; ROS, reactive oxygen species; circle P, phosphorylation.

Techniques Used: Activation Assay

The effect of mitochondrial Ca 2+ uptake inhibition on i.t. NMDA-induced protein kinase A (PKA) or extracellular signal-related kinase (ERK) activation in spinal dorsal horn neurons. a–i , Images of superficial dorsal horn (laminae I–II) immunostained for pPKA in 3 groups of mice: normal ( a–c ), with i.t. NMDA ( d–f ) and with i.t. NMDA + Ru360 ( g–i ). Double-labeled cells with pPKA (red) and NeuN (green; neuronal marker) appear in yellow in merged panels ( c, f, i ). j . The averaged percentages of pPKA positive neurons (I–II). k–m , Images of superficial dorsal horn immunostained for pERK in normal ( k ), i.t. NMDA- ( l ) and i.t. NMDA + Ru360 ( m )-treated mice. The areas showing immunopositivity to pERK were quantified by densitometry in laminae I–II (within the dashed line area) and the data are shown in the graph n . Both PKA and ERK were activated by i.t. NMDA but this activation was significantly reduced by blocking mitochondrial Ca 2+ uptake. †P
Figure Legend Snippet: The effect of mitochondrial Ca 2+ uptake inhibition on i.t. NMDA-induced protein kinase A (PKA) or extracellular signal-related kinase (ERK) activation in spinal dorsal horn neurons. a–i , Images of superficial dorsal horn (laminae I–II) immunostained for pPKA in 3 groups of mice: normal ( a–c ), with i.t. NMDA ( d–f ) and with i.t. NMDA + Ru360 ( g–i ). Double-labeled cells with pPKA (red) and NeuN (green; neuronal marker) appear in yellow in merged panels ( c, f, i ). j . The averaged percentages of pPKA positive neurons (I–II). k–m , Images of superficial dorsal horn immunostained for pERK in normal ( k ), i.t. NMDA- ( l ) and i.t. NMDA + Ru360 ( m )-treated mice. The areas showing immunopositivity to pERK were quantified by densitometry in laminae I–II (within the dashed line area) and the data are shown in the graph n . Both PKA and ERK were activated by i.t. NMDA but this activation was significantly reduced by blocking mitochondrial Ca 2+ uptake. †P

Techniques Used: Inhibition, Activation Assay, Mouse Assay, Labeling, Marker, Blocking Assay

28) Product Images from "The Hexane Fraction of Bursera microphylla A. Gray Induces p21-Mediated Anti-Proliferative and Pro-Apoptotic Effects in Human Cancer-Derived Cell Lines"

Article Title: The Hexane Fraction of Bursera microphylla A. Gray Induces p21-Mediated Anti-Proliferative and Pro-Apoptotic Effects in Human Cancer-Derived Cell Lines

Journal: Integrative Cancer Therapies

doi: 10.1177/1534735417696721

Time-course effects of BM-H on MAPK pathway. Western blot bands represent phosphorylated ERK (p-ERK), total ERK (ERK), actin (as a housekeeping gene for ERK), phosphorylated p38 (p-p38), total p38 (p38), phosphorylated Akt (p-Akt), total Akt (Akt), and the housekeeping gene tubulin (to control both p-38 and Akt). Proteins were extracted from OCI cells treated with the vehicle (control) or with BM-H (BM-H) after 4, 8, 14, or 24 hours. Western blots are representative of 3 independent experiments.
Figure Legend Snippet: Time-course effects of BM-H on MAPK pathway. Western blot bands represent phosphorylated ERK (p-ERK), total ERK (ERK), actin (as a housekeeping gene for ERK), phosphorylated p38 (p-p38), total p38 (p38), phosphorylated Akt (p-Akt), total Akt (Akt), and the housekeeping gene tubulin (to control both p-38 and Akt). Proteins were extracted from OCI cells treated with the vehicle (control) or with BM-H (BM-H) after 4, 8, 14, or 24 hours. Western blots are representative of 3 independent experiments.

Techniques Used: Western Blot

29) Product Images from "TGF? induces GDNF responsiveness in neurons by recruitment of GFR?1 to the plasma membrane"

Article Title: TGF? induces GDNF responsiveness in neurons by recruitment of GFR?1 to the plasma membrane

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200203115

Pretreatment with TGF β induces GDNF responsiveness, but does not affect expression of the GDNF receptors. (A) 100,000 CG neurons per well were plated and allowed to attach for 2 h. The cells were treated with 2 ng/ml TGFβ for 3 h or left untreated as indicated. Subsequently no factor or GDNF (10 ng/ml) or GDNF (10 ng/ml) plus TGFβ (2 ng/ml) were added for 30 min before harvesting of the cells in Laemmli sample buffer. (B) CG neurons were handled as in (A) except that PD98059 or Wortmannin was added to prove specific inhibition of ERK- and Akt-phosphorylation by the respective inhibitors. (C) Cells were pretreated for 3 h with TGFβ (2 ng/ml), thereafter an antibody blocking all three TGFβ-isoforms (anti-pan TGFβ) was added where indicated. 10 min later GDNF (10 ng/ml), TGFβ (2 ng/ml) or both factors were added for 30 min and the cells subsequently harvested in Laemmli sample buffer. Protein extracts (A–C) were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes and probed with anti– phospho ERK-, anti–phospho Akt- or anti–GFRα1-antibodies. To control for equal loading the membrane stripped and probed with an antibody against nonphosphorylated ERK. (D) CG neurons were differently treated to investigate the requirement of persistent TGFβ or GDNF treatment for survival. Cells were pretreated for 3 h with TGFβ3 (2 ng/ml). Thereafter, either TGFβ was blocked by addition of a pan TGFβ antibody and GDNF (10 ng/ml) was added or only GDNF was added in the absence of the antibody. Similarly the neurons were pretreated with GDNF (10 ng/ml) for 3 h and after a washout of GDNF, TGFβ3 (2 ng/ml) was added. As controls, neurons were simultaneously treated with GDNF and TGFβ in the presence or absence of the pan TGFβ antibody. The surviving neurons were counted after 24 h, each bar represents the mean and standard deviation of two independent experiments with every condition tested in triplicate per assay (***, P ≤ 0.01). (E) RT-PCR of the GDNF-receptors GFRα1 and Ret and immunoblot of GFRα1 using total RNA and protein extracts, respectively, from CG neurons treated for 3 h with TGFβ or untreated controls. For the immunoblot Neuro2a cells stably expressing GFRα1 were used as positive control. (F) Anti phospho-ERK immunoblot with extracts from CG neurons, pretreated with TGFβ for the indicated time points and subsequently treated with GDNF for 30 min or left untreated. As control for equal loading an immunoblot with an antibody detecting nonphosphorylated ERK was used.
Figure Legend Snippet: Pretreatment with TGF β induces GDNF responsiveness, but does not affect expression of the GDNF receptors. (A) 100,000 CG neurons per well were plated and allowed to attach for 2 h. The cells were treated with 2 ng/ml TGFβ for 3 h or left untreated as indicated. Subsequently no factor or GDNF (10 ng/ml) or GDNF (10 ng/ml) plus TGFβ (2 ng/ml) were added for 30 min before harvesting of the cells in Laemmli sample buffer. (B) CG neurons were handled as in (A) except that PD98059 or Wortmannin was added to prove specific inhibition of ERK- and Akt-phosphorylation by the respective inhibitors. (C) Cells were pretreated for 3 h with TGFβ (2 ng/ml), thereafter an antibody blocking all three TGFβ-isoforms (anti-pan TGFβ) was added where indicated. 10 min later GDNF (10 ng/ml), TGFβ (2 ng/ml) or both factors were added for 30 min and the cells subsequently harvested in Laemmli sample buffer. Protein extracts (A–C) were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes and probed with anti– phospho ERK-, anti–phospho Akt- or anti–GFRα1-antibodies. To control for equal loading the membrane stripped and probed with an antibody against nonphosphorylated ERK. (D) CG neurons were differently treated to investigate the requirement of persistent TGFβ or GDNF treatment for survival. Cells were pretreated for 3 h with TGFβ3 (2 ng/ml). Thereafter, either TGFβ was blocked by addition of a pan TGFβ antibody and GDNF (10 ng/ml) was added or only GDNF was added in the absence of the antibody. Similarly the neurons were pretreated with GDNF (10 ng/ml) for 3 h and after a washout of GDNF, TGFβ3 (2 ng/ml) was added. As controls, neurons were simultaneously treated with GDNF and TGFβ in the presence or absence of the pan TGFβ antibody. The surviving neurons were counted after 24 h, each bar represents the mean and standard deviation of two independent experiments with every condition tested in triplicate per assay (***, P ≤ 0.01). (E) RT-PCR of the GDNF-receptors GFRα1 and Ret and immunoblot of GFRα1 using total RNA and protein extracts, respectively, from CG neurons treated for 3 h with TGFβ or untreated controls. For the immunoblot Neuro2a cells stably expressing GFRα1 were used as positive control. (F) Anti phospho-ERK immunoblot with extracts from CG neurons, pretreated with TGFβ for the indicated time points and subsequently treated with GDNF for 30 min or left untreated. As control for equal loading an immunoblot with an antibody detecting nonphosphorylated ERK was used.

Techniques Used: Expressing, Inhibition, Blocking Assay, SDS Page, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Positive Control

In the presence of a soluble GFR α 1 TGF β is not required to induce GDNF responsiveness. (A) CG neurons were seeded onto PORN/laminine-coated microtiter plates at a density of 3,000 cells per well. Cells were treated with combinations of 10 ng/ml GDNF, 2 ng/ml TGFβ3, soluble GFRα1 (sGFRα1, 100 nM), or anti-pan TGFβ antibody (10 μg/ml) as indicated. As a control the neurons were left untreated or CNTF (10 ng/ml) was added. (B) Immunoblot with a phospho-specific antibodies to Akt and ERK with extracts from CG neurons that have been either pretreated with TGFβ or a soluble GFRα1 for 3 h and where subsequently GDNF or GDNF plus TGFβ was added for 30 min.
Figure Legend Snippet: In the presence of a soluble GFR α 1 TGF β is not required to induce GDNF responsiveness. (A) CG neurons were seeded onto PORN/laminine-coated microtiter plates at a density of 3,000 cells per well. Cells were treated with combinations of 10 ng/ml GDNF, 2 ng/ml TGFβ3, soluble GFRα1 (sGFRα1, 100 nM), or anti-pan TGFβ antibody (10 μg/ml) as indicated. As a control the neurons were left untreated or CNTF (10 ng/ml) was added. (B) Immunoblot with a phospho-specific antibodies to Akt and ERK with extracts from CG neurons that have been either pretreated with TGFβ or a soluble GFRα1 for 3 h and where subsequently GDNF or GDNF plus TGFβ was added for 30 min.

Techniques Used:

30) Product Images from "The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4"

Article Title: The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4

Journal: BMB Reports

doi: 10.5483/BMBRep.2014.47.2.277

MAP1305 activates MAPKs in BMDC. (A) For MAPKs activation analysis, BMDCs were treated with MAP1305 (10 μg/ml) for 0, 5, 15, and 30 min, respectively. Cell lysates were prepared and immunoblotted with anti-p-p38, anti-p-JNK, anti-p-ERK, and anti-tubulin antibodies. The results are representative of 3 independent experiments. (B) BMDCs were pre-treated with SB203580 (10 μM), SP600125 (10 μM), and U0126 (5 μM) for 1 h, then cells were treated with or without MAP1305 (10 μg/ml) for 24 h. Levels of IL-6, TNF-α, and IL-1β were measured in triplicate, using ELISA. **P < 0.01; ***P < 0.001 compared with BMDC treated with MAP1305 only.
Figure Legend Snippet: MAP1305 activates MAPKs in BMDC. (A) For MAPKs activation analysis, BMDCs were treated with MAP1305 (10 μg/ml) for 0, 5, 15, and 30 min, respectively. Cell lysates were prepared and immunoblotted with anti-p-p38, anti-p-JNK, anti-p-ERK, and anti-tubulin antibodies. The results are representative of 3 independent experiments. (B) BMDCs were pre-treated with SB203580 (10 μM), SP600125 (10 μM), and U0126 (5 μM) for 1 h, then cells were treated with or without MAP1305 (10 μg/ml) for 24 h. Levels of IL-6, TNF-α, and IL-1β were measured in triplicate, using ELISA. **P < 0.01; ***P < 0.001 compared with BMDC treated with MAP1305 only.

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

31) Product Images from "A Klebsiella pneumoniae antibiotic resistance mechanism that subdues host defences and promotes virulence"

Article Title: A Klebsiella pneumoniae antibiotic resistance mechanism that subdues host defences and promotes virulence

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201607336

mgrB inactivation results in downregulation of early inflammatory responses in macrophages upon infection Immunoblot analysis of IκBα and tubulin levels in lysates of iBMDM cells infected with K. pneumoniae 52145, 52145‐Δ mgrB and 52145‐Δ mgrB Com for the indicated times. Immunoblot analysis of phospho‐ERK (P‐ERK), phospho‐p38 (P‐p38), phospho‐JNK (P‐JNK) and tubulin levels in lysates of iBMDMs cells infected with K. pneumoniae 52145, 52145‐Δ mgrB , 52145‐Δ mgrB Com and for the indicated times. Immunoblot analysis of phospho‐ERK (P‐ERK), phospho‐JNK (P‐JNK) and tubulin levels in lysates of iBMDMs cells infected with K. pneumoniae 52145, 52145‐Δ mgrB , 52145‐Δ pmrC ‐Δ lpxO ‐Δ mgrB ‐Δ pmrF ‐Δ pagP , 52145‐Δ mgrB ‐Δ pmrC , 52145‐Δ mgrB ‐Δ pagP , 52145‐Δ mgrB ‐Δ pmrF and 52145‐Δ mgrB ‐Δ lpxO for 40 min. TNF‐α secretion by iBMDM macrophages stimulated for 6 h with 1 × 10 5 UV‐killed K. pneumoniae 52145, 52145‐Δ mgrB , 52145‐Δ mgrB Com and 52145‐Δ pmrC ‐Δ lpxO ‐Δ mgrB ‐Δ pmrF ‐Δ pagP . *** P
Figure Legend Snippet: mgrB inactivation results in downregulation of early inflammatory responses in macrophages upon infection Immunoblot analysis of IκBα and tubulin levels in lysates of iBMDM cells infected with K. pneumoniae 52145, 52145‐Δ mgrB and 52145‐Δ mgrB Com for the indicated times. Immunoblot analysis of phospho‐ERK (P‐ERK), phospho‐p38 (P‐p38), phospho‐JNK (P‐JNK) and tubulin levels in lysates of iBMDMs cells infected with K. pneumoniae 52145, 52145‐Δ mgrB , 52145‐Δ mgrB Com and for the indicated times. Immunoblot analysis of phospho‐ERK (P‐ERK), phospho‐JNK (P‐JNK) and tubulin levels in lysates of iBMDMs cells infected with K. pneumoniae 52145, 52145‐Δ mgrB , 52145‐Δ pmrC ‐Δ lpxO ‐Δ mgrB ‐Δ pmrF ‐Δ pagP , 52145‐Δ mgrB ‐Δ pmrC , 52145‐Δ mgrB ‐Δ pagP , 52145‐Δ mgrB ‐Δ pmrF and 52145‐Δ mgrB ‐Δ lpxO for 40 min. TNF‐α secretion by iBMDM macrophages stimulated for 6 h with 1 × 10 5 UV‐killed K. pneumoniae 52145, 52145‐Δ mgrB , 52145‐Δ mgrB Com and 52145‐Δ pmrC ‐Δ lpxO ‐Δ mgrB ‐Δ pmrF ‐Δ pagP . *** P

Techniques Used: Infection

32) Product Images from "Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-α"

Article Title: Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-α

Journal:

doi: 10.3748/wjg.v13.i13.1962

Effect of L. plantarum on the ERK pathway. Caco-2 cells were incubated with L. plantarum , TNF-α or L. plantarum plus TNF-α. Cell lysates were immunoblotted with antibodies against phosphorylated ERK and total ERK. L. plantarum inhibited
Figure Legend Snippet: Effect of L. plantarum on the ERK pathway. Caco-2 cells were incubated with L. plantarum , TNF-α or L. plantarum plus TNF-α. Cell lysates were immunoblotted with antibodies against phosphorylated ERK and total ERK. L. plantarum inhibited

Techniques Used: Incubation

33) Product Images from "Regulatory effects of saponins from Panax japonicus on colonic epithelial tight junctions in aging rats"

Article Title: Regulatory effects of saponins from Panax japonicus on colonic epithelial tight junctions in aging rats

Journal: Journal of Ginseng Research

doi: 10.1016/j.jgr.2016.12.011

Effects of SPJ on the expression of the MAPK signaling pathway in the colon of aging rats. (A) Expression of the MAPK signaling pathway in the colon of rats by Western blot. (B) Quantification of p-ERK/ERK, p-P38/P38, p-JNK/β-actin, and JNK/β-actin ratios. Data are expressed as mean ± SEM ( n = 6). * p
Figure Legend Snippet: Effects of SPJ on the expression of the MAPK signaling pathway in the colon of aging rats. (A) Expression of the MAPK signaling pathway in the colon of rats by Western blot. (B) Quantification of p-ERK/ERK, p-P38/P38, p-JNK/β-actin, and JNK/β-actin ratios. Data are expressed as mean ± SEM ( n = 6). * p

Techniques Used: Expressing, Western Blot

34) Product Images from "Fas Signal Promotes the Immunosuppressive Function of Regulatory Dendritic Cells via the ERK/β-Catenin Pathway *"

Article Title: Fas Signal Promotes the Immunosuppressive Function of Regulatory Dendritic Cells via the ERK/β-Catenin Pathway *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.425751

TGF-β-induced ERK activation is responsible for the increased Fas expression of regulatory DCs. A , confocal analysis of Fas expression by regulatory DCs. Representative images of immunofluorescence were stained with CD11c-FITC, Fas-phycoerythrin,
Figure Legend Snippet: TGF-β-induced ERK activation is responsible for the increased Fas expression of regulatory DCs. A , confocal analysis of Fas expression by regulatory DCs. Representative images of immunofluorescence were stained with CD11c-FITC, Fas-phycoerythrin,

Techniques Used: Activation Assay, Expressing, Immunofluorescence, Staining

ERK overactivation-induced GSK-3 inactivation increases β-catenin expression in regulatory DCs. A , 1 × 10 6 imDCs and regulatory DCs were treated with 1 μg/ml Jo2 for 0–120 min and then lysed. The phosphorylation of ERK
Figure Legend Snippet: ERK overactivation-induced GSK-3 inactivation increases β-catenin expression in regulatory DCs. A , 1 × 10 6 imDCs and regulatory DCs were treated with 1 μg/ml Jo2 for 0–120 min and then lysed. The phosphorylation of ERK

Techniques Used: Expressing

35) Product Images from "Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-?B and proinflammatory gene program activation in intestinal epithelial cells"

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-?B and proinflammatory gene program activation in intestinal epithelial cells

Journal: BMC Microbiology

doi: 10.1186/1471-2180-4-33

Purified flagellin activates signaling pathways and proinflammatory gene expression in intestinal epithelial cells mimicking that of wildtype a wild-type Salmonella infection. HT29 cells were left untreated or treated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20 μg/ml)/PMA (12.5 ng/ml) for 10 min, or with flagellin (1 μg/ml) for the indicated times. WCE were prepared and analyzed by EMSA for NF-κB DNA binding activity, immuno-kinase assays (KA) or immunoblot analysis using phospho-specific antibodies for ERK or p38 to detect activation and with kinase-specific antibodies as described in Fig. 5A to detect bulk kinase abundance as indicated. A, EMSA to detect NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (α p65), normal rabbit serum (NRS) served as an irrelevant antibody control. B, immunoblot and kinase assays to detect IKK, JNK, ERK and p38 kinase activities and protein abundance as in Fig. 5A. C, semi-quantitative RT-PCR of proinflammatory gene expression of non-treated, wild-type and flagellin double mutant Salmonella typhimurium infected, TNFα (10 ng/ml) or flagellin (1 μg/ml) stimulated cells. HT29 cells were harvested at the indicated times after the indicated treatments and isolated RNA was used to make first strand cDNA that subsequently used in RT-PCR reactions (as described in Experimental Procedures) using gene-specific primers for IL1α, IL1β, IL-8, TNFα, MCP1 and β-actin. β-actin was used as a standard for normalizing expression patterns. Resulting PCR products were fractionated on 2% agarose gels and visualized by eithidium bromide staining.
Figure Legend Snippet: Purified flagellin activates signaling pathways and proinflammatory gene expression in intestinal epithelial cells mimicking that of wildtype a wild-type Salmonella infection. HT29 cells were left untreated or treated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20 μg/ml)/PMA (12.5 ng/ml) for 10 min, or with flagellin (1 μg/ml) for the indicated times. WCE were prepared and analyzed by EMSA for NF-κB DNA binding activity, immuno-kinase assays (KA) or immunoblot analysis using phospho-specific antibodies for ERK or p38 to detect activation and with kinase-specific antibodies as described in Fig. 5A to detect bulk kinase abundance as indicated. A, EMSA to detect NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (α p65), normal rabbit serum (NRS) served as an irrelevant antibody control. B, immunoblot and kinase assays to detect IKK, JNK, ERK and p38 kinase activities and protein abundance as in Fig. 5A. C, semi-quantitative RT-PCR of proinflammatory gene expression of non-treated, wild-type and flagellin double mutant Salmonella typhimurium infected, TNFα (10 ng/ml) or flagellin (1 μg/ml) stimulated cells. HT29 cells were harvested at the indicated times after the indicated treatments and isolated RNA was used to make first strand cDNA that subsequently used in RT-PCR reactions (as described in Experimental Procedures) using gene-specific primers for IL1α, IL1β, IL-8, TNFα, MCP1 and β-actin. β-actin was used as a standard for normalizing expression patterns. Resulting PCR products were fractionated on 2% agarose gels and visualized by eithidium bromide staining.

Techniques Used: Purification, Expressing, Infection, Binding Assay, Activity Assay, Activation Assay, Quantitative RT-PCR, Mutagenesis, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

36) Product Images from "Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration"

Article Title: Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001981

Differential activation of MAP kinases in O. tsutsugamushi -infected DCs upon exposure to CCL19. DCs were stimulated with O. tsutsugamushi (OT), or LPS for 18 h and then further incubated with CCL19 (200 ng/ml) for the indicated times. The activation of ERK and p38 MAP kinases was assessed by immunoblot using specific anti-phospho-ERK1/2 or phospho-p38 MAP kinases antibodies. ERK1/2, p38, and GAPDH were used as loading controls. CNT: immature DCs.
Figure Legend Snippet: Differential activation of MAP kinases in O. tsutsugamushi -infected DCs upon exposure to CCL19. DCs were stimulated with O. tsutsugamushi (OT), or LPS for 18 h and then further incubated with CCL19 (200 ng/ml) for the indicated times. The activation of ERK and p38 MAP kinases was assessed by immunoblot using specific anti-phospho-ERK1/2 or phospho-p38 MAP kinases antibodies. ERK1/2, p38, and GAPDH were used as loading controls. CNT: immature DCs.

Techniques Used: Activation Assay, Infection, Incubation

37) Product Images from "Palmitoylation-dependent Estrogen Receptor ? Membrane Localization: Regulation by 17?-Estradiol"

Article Title: Palmitoylation-dependent Estrogen Receptor ? Membrane Localization: Regulation by 17?-Estradiol

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-07-0547

Effect of ERα palmitoylation on E2-induced ERK and AKT phosphorylation. Western blot analysis of ERK phosphorylation in ERα (lanes 1-4) or ERα-Cys447Ala mutant (lanes 5 and 6)-transfected HeLa and HepG2 (lanes 7-9) cells were performed
Figure Legend Snippet: Effect of ERα palmitoylation on E2-induced ERK and AKT phosphorylation. Western blot analysis of ERK phosphorylation in ERα (lanes 1-4) or ERα-Cys447Ala mutant (lanes 5 and 6)-transfected HeLa and HepG2 (lanes 7-9) cells were performed

Techniques Used: Western Blot, Mutagenesis, Transfection

38) Product Images from "Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury"

Article Title: Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury

Journal: Biochemical pharmacology

doi: 10.1016/j.bcp.2005.12.028

Berberine blocked MEK/ERK activation, as well as Egr-1 and c-Fos expression induced by injury. SMCs were injured in the absence or in the presence of berberine (120 μM): (A) 10 min post-injury, the levels of phosphorylated MEK and ERK were determined; (B) 90 min post-injury, the levels of Egr-1 and c-Fos proteins were measured.
Figure Legend Snippet: Berberine blocked MEK/ERK activation, as well as Egr-1 and c-Fos expression induced by injury. SMCs were injured in the absence or in the presence of berberine (120 μM): (A) 10 min post-injury, the levels of phosphorylated MEK and ERK were determined; (B) 90 min post-injury, the levels of Egr-1 and c-Fos proteins were measured.

Techniques Used: Activation Assay, Expressing

Mechanical injury-stimulated Egr-1 and c-Fos expression was MEK/ERK-dependent. SMCs were diffusely injured with 64 lines of scraping by sterile fork tips. Cell lysates were isolated: (A) the levels of Egr-1 and c-Fos protein, and (B) the levels of ERK, phosphorylated ERK, MEK, and phospho-MEK were detected by Western blot analysis. (C) U0126 blocked injury-induced MEK/ERK activation as well as Egr-1 and c-Fos expression. SMCs were treated with a specific MEK inhibitor, U0126 (20 μM) 30 min prior to mechanical injury. Cell lysates were isolated; the levels of MEK/ERK, phosphor-MEK/ERK, Egr-1 and c-Fos were measured by Western blot analysis.
Figure Legend Snippet: Mechanical injury-stimulated Egr-1 and c-Fos expression was MEK/ERK-dependent. SMCs were diffusely injured with 64 lines of scraping by sterile fork tips. Cell lysates were isolated: (A) the levels of Egr-1 and c-Fos protein, and (B) the levels of ERK, phosphorylated ERK, MEK, and phospho-MEK were detected by Western blot analysis. (C) U0126 blocked injury-induced MEK/ERK activation as well as Egr-1 and c-Fos expression. SMCs were treated with a specific MEK inhibitor, U0126 (20 μM) 30 min prior to mechanical injury. Cell lysates were isolated; the levels of MEK/ERK, phosphor-MEK/ERK, Egr-1 and c-Fos were measured by Western blot analysis.

Techniques Used: Expressing, Isolation, Western Blot, Activation Assay

39) Product Images from "Effects of Oleacein on High-Fat Diet-Dependent Steatosis, Weight Gain, and Insulin Resistance in Mice"

Article Title: Effects of Oleacein on High-Fat Diet-Dependent Steatosis, Weight Gain, and Insulin Resistance in Mice

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2018.00116

Effects of oleacein on insulin levels, liver steatosis, and Western blot analyses of SREBP-1 and ERK. (A) Hematoxylin and eosin staining of liver sections (20× magnification). Liver of obese mice fed with high-fat diet (HFD) was rich in fat vacuoles (indicated by the arrows). For steatosis evaluation, a numerical score was assigned to each section, on the basis of the percentage of vacuolated cells as calculated for each mouse liver: 1 = 1–25%, 2 = 30–65%, 3 = > 70%. (B) Protein expression levels of SREBP-1, ERK, and p-ERK were detected in liver from oleacein-treated and untreated mice fed with HFD and mice fed with NCD. GAPDH, control of protein loading. (C) Blood samples were collected as indicated in the Section “ Materials and Methods .” Insulin levels were measured after 13 weeks of feeding with HFD with and without oleacein. Values are expressed as mean ± SD. * p
Figure Legend Snippet: Effects of oleacein on insulin levels, liver steatosis, and Western blot analyses of SREBP-1 and ERK. (A) Hematoxylin and eosin staining of liver sections (20× magnification). Liver of obese mice fed with high-fat diet (HFD) was rich in fat vacuoles (indicated by the arrows). For steatosis evaluation, a numerical score was assigned to each section, on the basis of the percentage of vacuolated cells as calculated for each mouse liver: 1 = 1–25%, 2 = 30–65%, 3 = > 70%. (B) Protein expression levels of SREBP-1, ERK, and p-ERK were detected in liver from oleacein-treated and untreated mice fed with HFD and mice fed with NCD. GAPDH, control of protein loading. (C) Blood samples were collected as indicated in the Section “ Materials and Methods .” Insulin levels were measured after 13 weeks of feeding with HFD with and without oleacein. Values are expressed as mean ± SD. * p

Techniques Used: Western Blot, Staining, Mouse Assay, Expressing

40) Product Images from "Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis *"

Article Title: Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.373159

MMP-13-promoted angiogenesis is mediated by FAK and ERK signaling pathway. A , levels of total and phosphorylated forms of FAK, Src, and ERK after treatment of HuhT1 cells with MMP-13 (100 ng/ml) shown by Western blotting. β-Actin expression was
Figure Legend Snippet: MMP-13-promoted angiogenesis is mediated by FAK and ERK signaling pathway. A , levels of total and phosphorylated forms of FAK, Src, and ERK after treatment of HuhT1 cells with MMP-13 (100 ng/ml) shown by Western blotting. β-Actin expression was

Techniques Used: Western Blot, Expressing

Related Articles

other:

Article Title: Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML
Article Snippet: Additional antibodies include: anti-phosphotyrosine 4G10 (Millipore, Darmstadt, Germany), anti-phospho AKT (Epitomics, Burlingame, CA, USA), anti-phospho ERK (Santa-Cruz Biotechnology Inc., Dallas, TX, USA), anti-phospho S6K and anti-S6K (Abcam, Cambridge, UK), anti-phospho p38 and anti-p38 (BD biosciences, Sparks, MD, USA) and anti-tubulin and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA).

Article Title: MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts
Article Snippet: Anti-phospho ERK, ERK2 and FAK (C-terminal) antibodies used for immunoblotting were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Mass Spectrometry:

Article Title: Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1
Article Snippet: .. Primary antibodies were used as follows: anti-IGFBP3 (MAB305, R & D Systems), anti-integrin β1 (Santa Cruz, Dallas, TX, USA), anti-α-tubulin (MS-581-P0, Thermo Scientific), anti-focal adhesion kinase (FAK, sc-557, Santa Cruz), anti-phosphorylated FAK (611806, BD), anti-Src (#2109, Cell Signaling, Beverly, MA, USA), anti-phosphorylated Src (05–677, Millipore), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB, sc-8008, Santa Cruz), anti-phosphorylated NF-kB (#3033, Cell Signaling), anti-protein kinase B/AKT (#9272, Cell Signaling), anti-phosphorylated AKT (#9271, Cell Signaling), anti-ERK (sc-94, Santa Cruz). anti-phospho-ERK (sc-7383, Santa Cruz), anti-integrin-linked kinase (ILK, GTX101691, GeneTex, Irvine, CA, USA), MMP1 (RB1536P0, Thermo Scientific) and MMP10 (AP6194a, Abgent, San Diego, CA, USA). .. Enzyme-linked immunosorbemt assay (ELISA) This study was approved by the institutional review boards of the National Cheng Kung University Hospital and the National Health Research Institutes.

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    Santa Cruz Biotechnology anti phospho erk
    Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The <t>4G10</t> (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho <t>ERK</t> and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.
    Anti Phospho Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti phospho extracellular signal regulated kinase erk
    Effects of the PAR1 antagonist SCH79797 (SCH) and the PI 3 K/Akt inhibitor LY29004 (LY) on <t>ERK,</t> phospho-ERK and c-Jun as assessed by western blot assay after transient global cerebral I/R injury. (A) Representative immunoblots of ERK, phospho-ERK, and c-Jun in hippocampal tissue homogenates from the studied groups 48 hours after I/R. (B) Densitometry analysis results for ERK phosphorylation and c-Jun 48 hours after I/R. The graph shows optical density ratios of the target protein to <t>β-actin</t> using data combined from three independent experiments after normalization to the loading control. Sham group: Sham-operated control rabbits; saline group: rabbits subjected to 3 minutes of cardiac arrest and administered saline; SCH group: rabbits subjected to 3 minutes of cardiac arrest and treated with PAR1 receptor antagonist SCH79797 (25 μg/kg, intravenously) 10 minutes after cardiopulmonary resuscitation; LY + SCH group: rabbits that received the PI 3 K/Akt inhibitor LY29004 (3 mg/kg, intravenously) 10 minutes before SCH administration. Data are expressed as the mean ± SD. Differences among groups were analyzed by one-way analysis of variance followed by Bonferroni post hoc tests. ** P
    Anti Phospho Extracellular Signal Regulated Kinase Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

    Journal: Oncogene

    Article Title: Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML

    doi: 10.1038/onc.2016.41

    Figure Lengend Snippet: Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

    Article Snippet: Additional antibodies include: anti-phosphotyrosine 4G10 (Millipore, Darmstadt, Germany), anti-phospho AKT (Epitomics, Burlingame, CA, USA), anti-phospho ERK (Santa-Cruz Biotechnology Inc., Dallas, TX, USA), anti-phospho S6K and anti-S6K (Abcam, Cambridge, UK), anti-phospho p38 and anti-p38 (BD biosciences, Sparks, MD, USA) and anti-tubulin and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Concentration Assay, Cell Culture, Immunoprecipitation, SDS Page, Western Blot

    Fig. 7. Fibroblast migration and calpain activity are dependent on MEK, but not JNK activity. ( A ) Wild-type fibroblasts were loaded into Transwell migration chambers (10 5 cells/well) and allowed to migrate for 5 h, using 5% serum as a chemotactic agent. Calpain inhibitor PD150606 (50 µM), MEK inhibitor UO126 (10 µM) or matrix metalloproteinase inhibitor GM6001 (2 µM) were added to both the upper and lower chambers of the designated wells. ( B ) Wild-type fibroblasts were pre-incubated for 1 h with JNK inhibitor SP600125 (10 µM) or MEK inhibitor UO126 (10 µM), and calpain activity was assessed by SLLVY-AMC cleavage, and compared with that of non-treated cells. The results of both (A) and (B) are the mean ± SEM of at least three independent experiments. ( C ) Wild-type fibroblasts were pre-incubated with 10 µM SP600125 or 10 µM UO126 for 1 h and then analyzed for migration using the in vitro wound healing assay in the continuous presence of inhibitor. ( D ) Serum-starved wild-type, MEKK1–/– or MEKK1 add-back fibroblasts were treated with EGF or FGF-2 for 10 min and then lysed. ERK1/2 activation was then assessed by phospho-ERK immunoblotting. The membrane was then stripped and the total ERK2 level determined by ERK2 immunoblotting. The data are representative of at least three independent experiments. NS, no stimulus.

    Journal: The EMBO Journal

    Article Title: MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts

    doi: 10.1093/emboj/cdg322

    Figure Lengend Snippet: Fig. 7. Fibroblast migration and calpain activity are dependent on MEK, but not JNK activity. ( A ) Wild-type fibroblasts were loaded into Transwell migration chambers (10 5 cells/well) and allowed to migrate for 5 h, using 5% serum as a chemotactic agent. Calpain inhibitor PD150606 (50 µM), MEK inhibitor UO126 (10 µM) or matrix metalloproteinase inhibitor GM6001 (2 µM) were added to both the upper and lower chambers of the designated wells. ( B ) Wild-type fibroblasts were pre-incubated for 1 h with JNK inhibitor SP600125 (10 µM) or MEK inhibitor UO126 (10 µM), and calpain activity was assessed by SLLVY-AMC cleavage, and compared with that of non-treated cells. The results of both (A) and (B) are the mean ± SEM of at least three independent experiments. ( C ) Wild-type fibroblasts were pre-incubated with 10 µM SP600125 or 10 µM UO126 for 1 h and then analyzed for migration using the in vitro wound healing assay in the continuous presence of inhibitor. ( D ) Serum-starved wild-type, MEKK1–/– or MEKK1 add-back fibroblasts were treated with EGF or FGF-2 for 10 min and then lysed. ERK1/2 activation was then assessed by phospho-ERK immunoblotting. The membrane was then stripped and the total ERK2 level determined by ERK2 immunoblotting. The data are representative of at least three independent experiments. NS, no stimulus.

    Article Snippet: Anti-phospho ERK, ERK2 and FAK (C-terminal) antibodies used for immunoblotting were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Migration, Activity Assay, Incubation, In Vitro, Wound Healing Assay, Activation Assay

    Effects of PGSF on the phosphorylation of NR2B, CaMKII, ERK, and CREB in hippocampus of rats after LTP induction. Representative Western blotting of phosphorylated and total forms of NR2B, CaMKII, ERK, and CREB (A, C, E, and G). The histogram showed the densitometric quantitation of the immunoreactive bands at different times after LTP induction (B, D, F, and H). The phosphorylation levels of NR2B, CaMKII, ERK, and CREB were normalized by total NR2B, CaMKII, ERK, and CREB and expressed as fold changes of their respective controls (mean±SEM; n =6). b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Polygalasaponin F induces long-term potentiation in adult rat hippocampus via NMDA receptor activation

    doi: 10.1038/aps.2011.199

    Figure Lengend Snippet: Effects of PGSF on the phosphorylation of NR2B, CaMKII, ERK, and CREB in hippocampus of rats after LTP induction. Representative Western blotting of phosphorylated and total forms of NR2B, CaMKII, ERK, and CREB (A, C, E, and G). The histogram showed the densitometric quantitation of the immunoreactive bands at different times after LTP induction (B, D, F, and H). The phosphorylation levels of NR2B, CaMKII, ERK, and CREB were normalized by total NR2B, CaMKII, ERK, and CREB and expressed as fold changes of their respective controls (mean±SEM; n =6). b P

    Article Snippet: Anti-phospho-CaMKII antibody, anti-CaMKII antibody, anti-phospho-ERK antibody, anti-ERK antibody, and anti-rabbit IgG secondary antibody, anti-mouse IgG secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Western Blot, Quantitation Assay

    Effects of the PAR1 antagonist SCH79797 (SCH) and the PI 3 K/Akt inhibitor LY29004 (LY) on ERK, phospho-ERK and c-Jun as assessed by western blot assay after transient global cerebral I/R injury. (A) Representative immunoblots of ERK, phospho-ERK, and c-Jun in hippocampal tissue homogenates from the studied groups 48 hours after I/R. (B) Densitometry analysis results for ERK phosphorylation and c-Jun 48 hours after I/R. The graph shows optical density ratios of the target protein to β-actin using data combined from three independent experiments after normalization to the loading control. Sham group: Sham-operated control rabbits; saline group: rabbits subjected to 3 minutes of cardiac arrest and administered saline; SCH group: rabbits subjected to 3 minutes of cardiac arrest and treated with PAR1 receptor antagonist SCH79797 (25 μg/kg, intravenously) 10 minutes after cardiopulmonary resuscitation; LY + SCH group: rabbits that received the PI 3 K/Akt inhibitor LY29004 (3 mg/kg, intravenously) 10 minutes before SCH administration. Data are expressed as the mean ± SD. Differences among groups were analyzed by one-way analysis of variance followed by Bonferroni post hoc tests. ** P

    Journal: Neural Regeneration Research

    Article Title: A protease-activated receptor 1 antagonist protects against global cerebral ischemia/reperfusion injury after asphyxial cardiac arrest in rabbits

    doi: 10.4103/1673-5374.199011

    Figure Lengend Snippet: Effects of the PAR1 antagonist SCH79797 (SCH) and the PI 3 K/Akt inhibitor LY29004 (LY) on ERK, phospho-ERK and c-Jun as assessed by western blot assay after transient global cerebral I/R injury. (A) Representative immunoblots of ERK, phospho-ERK, and c-Jun in hippocampal tissue homogenates from the studied groups 48 hours after I/R. (B) Densitometry analysis results for ERK phosphorylation and c-Jun 48 hours after I/R. The graph shows optical density ratios of the target protein to β-actin using data combined from three independent experiments after normalization to the loading control. Sham group: Sham-operated control rabbits; saline group: rabbits subjected to 3 minutes of cardiac arrest and administered saline; SCH group: rabbits subjected to 3 minutes of cardiac arrest and treated with PAR1 receptor antagonist SCH79797 (25 μg/kg, intravenously) 10 minutes after cardiopulmonary resuscitation; LY + SCH group: rabbits that received the PI 3 K/Akt inhibitor LY29004 (3 mg/kg, intravenously) 10 minutes before SCH administration. Data are expressed as the mean ± SD. Differences among groups were analyzed by one-way analysis of variance followed by Bonferroni post hoc tests. ** P

    Article Snippet: The blots were blocked in 5% dry skim milk and then incubated at 4°C overnight with primary antibodies of mouse origin, including anti-phospho-extracellular signal-regulated kinase (ERK), anti-ERK, anti-c-Jun, and anti-β-actin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) as well as anti-procaspase 3 and anti-cleaved caspase 3 (1:500; Sigma, St. Louis, MO, USA).

    Techniques: Western Blot