Journal: International Journal of Biomedical Science : IJBS

Article Title: Role of Raf-1 Kinase in Diabetes-Induced Accelerated Apoptosis of Retinal Capillary Cells

doi:

Figure Lengend Snippet: Raf-1 kinase modulates glucose-induced apoptosis of retinal endothelial cells. Apoptosis was measured by performing ELISA for cytoplasmic histone-associated-DNA-fragments using an assay kit from Roche Diagnostics. The figure shows the values obtained from the cells incubated with glucose for five days, and these values were adjusted to the total DNA. The values obtained with 5 mM glucose were considered 100%. 5 MmGlu, 5 mM glucose; 20 mMGlu, 20 mM glucose; +GW5074=20 mM glucose+10 μM GW5074; +ZM336374=20 mM glucose+200 μM ZM336374; +PD098059=20 mM glucose+30 μM PD098059. * P <0.05 compared to 5 mM glucose and # P <0.05 compared to 20 mM glucose.

Article Snippet: To specifically investigate the effect of MEK pathway in accelerated apoptosis of endothelial cells, we incubated the cells with or without MEK inhibitor PD098059 (2’-amino-3’methoxyflavone, United States Biological, Swampscott, MA) at a final concentration of 30 μM ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: International Journal of Biomedical Science : IJBS

Article Title: Role of Raf-1 Kinase in Diabetes-Induced Accelerated Apoptosis of Retinal Capillary Cells

doi:

Figure Lengend Snippet: Raf-1 kinase and MEK regulate NF-kB activation: Gene expression of NF- κ B was determined by Q-RT-PCR using Taqman primers and probe designed by the use of File Builder 3.0 (ABI). The results are mean ± SD of three of more experiments with each measurement made in triplicate. 5 mMGlu, 5 mM glucose; 20 mMGlu, 20 mM glucose; +GW5074=20 mM glucose+10 μM GW5074; +ZM336374=20 mM glucose+200 μM ZM336374; +PD098059=20 mM glucose+30 μM PD098059. * P <0.05 compared to 5 mM glucose and # P <0.05 compared to 20 mM glucose.

Article Snippet: To specifically investigate the effect of MEK pathway in accelerated apoptosis of endothelial cells, we incubated the cells with or without MEK inhibitor PD098059 (2’-amino-3’methoxyflavone, United States Biological, Swampscott, MA) at a final concentration of 30 μM ( ).

Techniques: Activation Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Biomedical Science : IJBS

Article Title: Role of Raf-1 Kinase in Diabetes-Induced Accelerated Apoptosis of Retinal Capillary Cells

doi:

Figure Lengend Snippet: Caspase-3 is implicated in Raf-1 mediated glucose-induced increased apoptosis of retinal endothelial cells. Activity of apoptosis execution enzyme- caspase-3 was determined in the cells by measuring the cleavage of Ac-DEVD-pNA at 405 nm. The values obtained from the cells incubated in 5 mM glucose are considered 100%. Each experiment was repeated with 3-4 cell preparations, and measurements done in duplicate. 5 mMGlu, 5 mM glucose; 20 mMGlu, 20 mM glucose; +GW5074=20 mM glucose+10 μM GW5074; +ZM336374=20 mM glucose+200 μM ZM336374; +PD098059=20 mM glucose+30 μM PD098059. * P <0.05 compared to 5 mM glucose and # P <0.05 compared to 20 mM glucose.

Article Snippet: To specifically investigate the effect of MEK pathway in accelerated apoptosis of endothelial cells, we incubated the cells with or without MEK inhibitor PD098059 (2’-amino-3’methoxyflavone, United States Biological, Swampscott, MA) at a final concentration of 30 μM ( ).

Techniques: Activity Assay, Incubation

Journal: The Journal of Clinical Investigation

Article Title: Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer

doi: 10.1172/JCI167651

Figure Lengend Snippet: ( A ) Bar graph depicting fold change in cell numbers in NSCLC cell lines harboring various mutations in KRAS treated with vehicle (DMSO), 25 nM eFT226 (eIF4Ai), 50 nM trametinib (MEKi), or both agents together (Combo) for 72 hours in 2D/low-serum conditions. Data are represented as means ± SD. n = 3. *** P < 0.001; **** P < 0.0001, 1-way ANOVA and Tukey’s post hoc test. H23 ( KRAS G12C ), H1573 ( KRAS G12A ), LU99 ( KRAS G12C ), H1792 ( KRAS G12C ), HCC44 ( KRAS G12C ), LU65 ( KRAS G12C ), H441 ( KRAS G12V ), H1944 ( KRAS G13D ), H2122 ( KRAS G12C ), A549 ( KRAS G12S ), H1373 ( KRAS G12C ), H2030 ( KRAS G12C ), H1355 ( KRAS G13C ). ( B ) Immunoblots for each cell line showing suppression of phospho-ERK by trametinib (MEKi) after 24 hours. ( C ) Bar graphs depicting fold change in cell numbers of specified cell lines transfected with either siCNT or siEIF4A1 and treated with vehicle (DMSO) or 50 nM trametinib (MEKi) for 72 hours in 2D/low-serum conditions. Data are represented as means ± SD. n = 3. **** P < 0.0001, 1-way ANOVA and Tukey’s post hoc test. Immunoblots confirm suppression of eIF4A1 by siRNAs.

Article Snippet: For drug treatments, eIF4A inhibitor eFT226 and KRAS G12C inhibitor MRTX849 were purchased from MedChemExpress; MEK inhibitor trametinib was purchased from LC Laboratories; BCL-xL/BCL-2 inhibitor navitoclax, BCL-2 inhibitor venetoclax, and CDK4/6 inhibitor palbociclib were purchased from Selleckchem; and MCL1 inhibitor S63845 was purchased from Chemietek.

Techniques: Western Blot, Transfection

Journal: The Journal of Clinical Investigation

Article Title: Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer

doi: 10.1172/JCI167651

Figure Lengend Snippet: ( A and B ) Synergy plots depicting the effects of indicated drug combinations using the HSA model. ( C and D ) Long-term cell proliferation assay of KRAS -mutant NSCLC cells treated with vehicle (DMSO), 25 nM eFT226 (eIF4Ai), 100 nM MRTX849 (KRASi), 50 nM trametinib (MEKi), or drug combinations (Combo) up to 3 weeks. ( E – G ) Incucyte live-cell imaging data depicting cleaved caspase-3/7 activity in sensitive H23 and H1573 and resistant H2030 cell lines in response to vehicle (DMSO), 25 nM eFT226 (eIF4Ai), 100 nM MRTX849 (KRASi), 50 nM trametinib (MEKi), or drug combinations (Combo).

Article Snippet: For drug treatments, eIF4A inhibitor eFT226 and KRAS G12C inhibitor MRTX849 were purchased from MedChemExpress; MEK inhibitor trametinib was purchased from LC Laboratories; BCL-xL/BCL-2 inhibitor navitoclax, BCL-2 inhibitor venetoclax, and CDK4/6 inhibitor palbociclib were purchased from Selleckchem; and MCL1 inhibitor S63845 was purchased from Chemietek.

Techniques: Proliferation Assay, Mutagenesis, Live Cell Imaging, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer

doi: 10.1172/JCI167651

Figure Lengend Snippet: Graphs depicting the fold change in tumor volume of ( A and B ) H23-derived xenograft models and ( C and D ) DFCI-730 PDX models treated for 28 days with vehicle, 100 mg/kg QD MRTX849 (KRASi), 0.5 mg/kg Q4D eFT226 (eIF4Ai), or the 2 agents together (Combo). Data are represented as means ± SEM. n = 7–8 tumors per condition. ( B and D ) Waterfall plots depicting fold change of each tumor within the 4 treatment arms after 28 days of treatment (versus day 0). **** P < 0.0001, 1-way ANOVA and Tukey’s post hoc test. Single asterisks indicate maximum tumor volume. These mice reached end point at days 21 and 25. Graphs depicting the fold change in tumor volume of ( E and F ) H1573-derived xenograft models and ( G and H ) H441-derived xenograft models treated for 28 days with vehicle, 0.6 mg/kg QD trametinib (MEKi), 0.5 mg/kg Q4D eFT226 (eIF4Ai), or the 2 agents together (Combo). Data are represented as means ± SEM. n = 7–10 tumors per condition. ( F and H ) Waterfall plots depicting fold change of each tumor within the 4 treatment arms after 28 days of treatment (versus day 0). **** P < 0.0001, 1-way ANOVA and Tukey’s post hoc test. Single asterisks indicate maximum tumor volume. These mice reached end point at days 18 and 21.

Article Snippet: For drug treatments, eIF4A inhibitor eFT226 and KRAS G12C inhibitor MRTX849 were purchased from MedChemExpress; MEK inhibitor trametinib was purchased from LC Laboratories; BCL-xL/BCL-2 inhibitor navitoclax, BCL-2 inhibitor venetoclax, and CDK4/6 inhibitor palbociclib were purchased from Selleckchem; and MCL1 inhibitor S63845 was purchased from Chemietek.

Techniques: Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer

doi: 10.1172/JCI167651

Figure Lengend Snippet: ( A ) Bar graphs depicting the effects of either KRASi (left, middle) or MEKi (right) in sensitive cell lines in the presence of the indicated siRNA pools. The fold change in cell number was calculated after 72 hours of treatment (versus day 0) in response to 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. The siCDK4 studies were performed separately; however, control values were similar (primary data are shown in ). *Complete genetic ablation of MCL1 alone in H23 cells resulted in cell death, preventing further analysis. ( B ) Bar graphs depicting fold changes in cell numbers in cells treated for 72 hours (versus day 0) with 100 nM S63845 (MCL1i) or 1 μM navitoclax (BCL-xL/BCL-2i) or 1 μM venetoclax (BCL-2i) combined with either 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. *** P < 0.001; **** P < 0.0001, 1-way ANOVA and Tukey’s post hoc test. ( C ) Bar graphs depicting fold change in cell number in cells treated for 72 hours with 500 nM palbociclib (CDK4/6i) combined with either 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. ** P < 0.01; *** P < 0.001; **** P < 0.0001, 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: For drug treatments, eIF4A inhibitor eFT226 and KRAS G12C inhibitor MRTX849 were purchased from MedChemExpress; MEK inhibitor trametinib was purchased from LC Laboratories; BCL-xL/BCL-2 inhibitor navitoclax, BCL-2 inhibitor venetoclax, and CDK4/6 inhibitor palbociclib were purchased from Selleckchem; and MCL1 inhibitor S63845 was purchased from Chemietek.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer

doi: 10.1172/JCI167651

Figure Lengend Snippet: ( A ) MYC copy-number variations (CNV) and KRAS mutational data of the 13 NSCLC cell lines retrieved from cBioPortal. ( B ) Immunoblots showing protein expression of MYC in NSCLC cells under baseline conditions. ( C ) GSEA analysis comparing the expression of MYC-regulated ribosomal and translation components in NSCLC cell lines at baseline. Heatmap shows expression of individual genes. ( D ) (Top) Bar graph depicting fold change in cell number of resistant NSCLC cells ectopically expressing control or MYC cDNAs treated for 72 hours with vehicle (DMSO) or combined 25 nM eFT226 (eIF4Ai) and either 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. **** P < 0.001, 1-way ANOVA and Tukey’s post hoc test. (Bottom) Immunoblots showing suppression of phospho-ERK and overexpression of MYC in response to specified treatments and MYC ectopic expression. o/e, overexpression. ( E ) (Left) Percentage live/dead of high MYC (DFCI-730, DFCI-456, MGH-9029-1B) and low MYC (MGH-1112-1, MGH-1196-2) PDOTSs treated with DMSO, 25 nM eFT226 (eIF4Ai), 100 nM MRTX849 (KRASi), or combined drugs for 6 days in 3D microfluidic culture. (Right) Immunoblots showing protein levels of MYC in PDX tumor samples under baseline conditions.

Article Snippet: For drug treatments, eIF4A inhibitor eFT226 and KRAS G12C inhibitor MRTX849 were purchased from MedChemExpress; MEK inhibitor trametinib was purchased from LC Laboratories; BCL-xL/BCL-2 inhibitor navitoclax, BCL-2 inhibitor venetoclax, and CDK4/6 inhibitor palbociclib were purchased from Selleckchem; and MCL1 inhibitor S63845 was purchased from Chemietek.

Techniques: Western Blot, Expressing, Over Expression

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: ( a ) Representative western blot of forebrain protein extracts from mice treated with or without 2% sevoflurane for 6 h at P6 (sevoflurane (−) (control): n = 6; sevoflurane (+): n = 6). ( b ) Cleaved PARP levels significantly increased in sevoflurane-treated mice compared to the levels of cleaved PARP in controls. ( c,d ) Expression levels of ERK1 and ERK2 in sevoflurane-treated mice were not significantly affected compared to those of the controls. ( e,f ) ERK1 and ERK2 phosphorylation levels significantly decreased in sevoflurane-treated mice compared to those of the controls. ( g ) Representative western blot of forebrain protein extracts from mice treated with 50 mg/kg of SL327 or vehicle (DMSO) at P6 (vehicle: n = 6; SL327: n = 6). ( h ) Cleaved PARP levels significantly increased in SL327-treated mice compared to the levels of cleaved PARP in vehicle controls. ( i,j ) Expression levels of ERK1 and ERK2 were not significantly affected in SL327-treated mice compared to that in vehicle controls. ( k,l ) ERK1 and ERK2 phosphorylation levels significantly decreased in SL327-treated mice compared to their expression levels in vehicle controls. To evaluate the levels of protein expression and phosphorylation, band levels were divided according to their corresponding internal loading control (β-actin). Data are represented as mean ± SEM. *** P < 0.001.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Western Blot, Control, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: ( a ) Representative western blot of forebrain protein extracts from mice treated with or without 2% sevoflurane for 6 h at P14 (sevoflurane (−) (control): n = 6; sevoflurane (+): n = 6). ( b ) Cleaved PARP levels slightly increased in sevoflurane-treated mice compared to their levels in controls. ( c,d ) ERK1 and ERK2 expression levels in sevoflurane-treated mice were not significantly different compared to controls. ( e,f ) ERK1 and ERK2 phosphorylation levels slightly decreased in sevoflurane-treated mice compared to their levels in the controls. ( g ) Representative western blot of forebrain protein extracts from mice treated with 50 mg/kg SL327 or vehicle (DMSO) at P14 (vehicle: n = 6; SL327: n = 6). ( h ) Cleaved PARP levels were not significantly affected in SL327-treated mice compared to their expression levels in vehicle controls. ( i,j ) Expression levels of ERK1 and ERK2 in SL327-treated mice were not changed significantly compared with those of vehicle controls. ( k,l ) ERK1 and ERK2 phosphorylation levels significantly decreased in SL327-treated mice compared to their phosphorylation levels in vehicle controls. The protein expression and phosphorylation levels were calculated as described in . Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Western Blot, Control, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: ( a ) Representative western blot of forebrain protein extracts from mice treated with or without 2% sevoflurane for 6 h at P21 (sevoflurane (−) (control): n = 5; sevoflurane (+): n = 5). ( b ) Cleaved PARP levels were not significantly affected in sevoflurane-treated mice compared to their levels in controls. ( c,d ) ERK1 and ERK2 expression levels in sevoflurane-treated mice were not significantly affected compared to their expression levels in controls. ( e,f ) ERK1 and ERK2 phosphorylation levels slightly decreased in sevoflurane-treated mice compared to their levels in controls. ( g ) Representative western blot of forebrain protein extracts from mice treated with 50 mg/kg SL327 or vehicle (DMSO) at P21 (vehicle control: n = 5; SL327: n = 5). ( h ) Cleaved PARP levels were not significantly affected in SL327-treated mice compared to their levels in vehicle controls. ( i,j ) ERK1 and ERK2 expression levels in SL327-treated mice were not significantly affected compared to their expression levels in vehicle controls. ( k,l ) ERK1 and ERK2 phosphorylation levels significantly decreased in SL327-treated mice compared to their levels in vehicle controls. The protein expression and phosphorylation levels were calculated as described in . Data are represented as mean ± SEM. * P < 0.05, *** P < 0.001.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Western Blot, Control, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: Representative high-power images of the brains of mice treated with vehicle ( a – c ), sevoflurane ( a’ – c ’), or SL327 ( a” – c” ). ( a,a’,a” ) cortex, ( b,b’,b” ) retrosplenial cortex, ( c,c’,c” ) subiculum. Scale bars: 250 μm.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques:

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: Double immunostaining of activated caspase-3 with cellular markers for neurons ( a,a’ ), astrocytes ( b,b’ ), and oligodendrocytes ( c,c’ ). ( a–c ) Merged images indicate that activated caspase-3 signals were observed in neurons ( a ) and oligodendrocytes ( c ), but not in astrocytes ( b ), 6 h after sevoflurane administration. ( a’–c’ ) Similarly, activated caspase-3 signals were observed in neurons ( a’ ) and oligodendrocytes ( c’ ), but not in astrocytes ( b ), 6 h after SL327 injection. ( a,a’,b,b’ ) dorsal hippocampal commissure; ( c,c’ ) cortex. Scale bars: 50 μm.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Double Immunostaining, Injection

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: Arterial blood gas analysis.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques:

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: ( a ) Representative western blot of forebrain protein extracts from mice treated with or without 2% sevoflurane in the presence of SL327 or vehicle at P6 (SL327 (−), sevoflurane (−): n = 8; SL327 (+), sevoflurane (−): n = 10; SL327 (−), sevoflurane (+): n = 8; SL327 (+), sevoflurane (+): n = 7). ( b ) SL327 administration at P6 significant increased cleaved PARP levels compared to vehicle controls when only carrier gas was administered (SL327 (−), sevoflurane (−) vs. SL327 (+), sevoflurane (−)). Conversely, no significant differences were observed between the SL327 and vehicle control groups when 2% sevoflurane was administered (SL327 (−), sevoflurane (+) vs. SL327 (+), sevoflurane (+)). ( c ) Representative western blot of forebrain protein extracts from mice treated with or without 2% sevoflurane in the presence of SL327 or vehicle at P14 (SL327 (−), sevoflurane (−): n = 6; SL327 (+), sevoflurane (−): n = 6; SL327 (−), sevoflurane (+): n = 6; SL327 (+), sevoflurane (+): n = 6). ( d ) No significant differences were observed in the cleaved PARP levels among the groups. To evaluate protein expression and phosphorylation, band levels were divided according to their corresponding internal loading control (β-actin). Data are represented as mean ± SEM. *** P < 0.001.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Western Blot, Control, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: ( a ) No significant differences were observed in total distance traveled over 3 consecutive days in a new cage at 10 weeks of age between mice treated with sevoflurane at P6 and controls (control: n = 7; sevoflurane: n = 7). Over an additional 7 days, the sevoflurane group travelled significantly longer distances compared to the vehicle group. ( b ) A significant difference was observed in immobility time in the forced swim test between mice treated with sevoflurane at P6 and controls (control: n = 7; sevoflurane: n = 7). ( c ) Significant difference was observed in immobility time in the tail suspension test between mice treated with sevoflurane at P6 and controls (control: n = 7; sevoflurane: n = 7). ( d ) No significant differences were observed in total distance traveled over 3 consecutive days in a new cage at 10 weeks of age between mice treated with SL327 at P6 and vehicle controls (control: n = 7; sevoflurane: n = 8). Over an additional 7 days, the sevoflurane group travelled significantly longer distances travelled compared to the vehicle group. ( e ) A significant difference was observed in the immobility time in the forced swim test between mice treated with SL327 at P6 and vehicle controls (control: n = 7; sevoflurane: n = 7). ( f ) A significant difference was observed in the immobility time in the tail suspension test between mice treated with SL327 at P6 and vehicle controls (control: n = 7; sevoflurane: n = 7). Data are represented as mean ± SEM. * P < 0.05.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Control, Suspension

Journal: Scientific Reports

Article Title: Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

doi: 10.1038/srep21859

Figure Lengend Snippet: ( a–d ) Molecular hydrogen (H 2 ) gas, an effective antioxidant, decreased cleaved PARP levels caused by sevoflurane exposure at P6, concomitant with restored ERK phosphorylation levels. ( a ) Representative western blot of forebrain protein extracts from mice treated with or without 2% sevoflurane for 6 h in the presence of 1.3% hydrogen or not at P6 (sevoflurane (−), hydrogen (−): n = 5; sevoflurane (+), hydrogen (−): n = 5; sevoflurane (+), hydrogen (+): n = 5). ( b ) The increased levels of cleaved PARP after sevoflurane exposure were attenuated by hydrogen (1.3%) administration at P6. ( c,d ) The decreased ERK phosphorylation levels were restored by concomitant treatment with hydrogen (1.3%). ( e ) Representative western blot of forebrain protein extracts from mice treated with SL327 with or without hydrogen (SL327 (+), hydrogen (−): n = 5; SL327 (+), hydrogen (−): n = 6). ( f ) Representative western blot of forebrain protein extracts from mice treated with vehicle (DMSO) with or without hydrogen (SL327 (−), hydrogen (−): n = 5; SL327 (−), hydrogen (−): n = 5). To evaluate protein expression and phosphorylation, band levels were divided according to their corresponding internal loading control (β-actin). Data are represented as mean ± SEM. ** P < 0.01, *** P < 0.001.

Article Snippet: ERK phosphorylation is suppressed by the MEK inhibitor, SL327 (ENZO, Farmingdale, NY), which penetrates the blood-brain barrier .

Techniques: Phospho-proteomics, Western Blot, Expressing, Control