p38 mapk inhibitor sb202190  (Abcam)

 
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    SB 202190
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    Structured Review

    Abcam p38 mapk inhibitor sb202190
    Effect of <t>p38</t> <t>MAPK</t> silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.

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    1) Product Images from "p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells"

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17020159

    Effect of p38 MAPK silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.
    Figure Legend Snippet: Effect of p38 MAPK silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.

    Techniques Used: Incubation, Western Blot, Concentration Assay, Negative Control

    Effect of p38 MAPK overexpression, using transfection with a specific plasmid (Vector with p38 MAPK) (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells transfected with an empty vector (Empty vector) represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. The fact that the band of p38 MAPK in the control samples is not visible here resulted from a large difference in p38 MAPK content in control and transfected cells. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with plasmid DNA containing p38 MAPK (Vector with p38 MAPK) and cells incubated with empty plasmid DNA (empty vector).
    Figure Legend Snippet: Effect of p38 MAPK overexpression, using transfection with a specific plasmid (Vector with p38 MAPK) (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells transfected with an empty vector (Empty vector) represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. The fact that the band of p38 MAPK in the control samples is not visible here resulted from a large difference in p38 MAPK content in control and transfected cells. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with plasmid DNA containing p38 MAPK (Vector with p38 MAPK) and cells incubated with empty plasmid DNA (empty vector).

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Incubation, Western Blot, Concentration Assay

    Effect of 1 mM stearic acid (SA) (see “Materials and Methods”) on ( A ) cell growth and viability; ( B ) the level of cleaved PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis); ( C ) the level of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (p38 MAPK signaling pathway); and ( D ) the level of phospho-c-Raf, phospho-MEK1/2, ERK1/2, phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without SA represented control cells. After 18 h of incubation (see “Materials and Methods”) for markers of apoptosis ( B ) and 3, 6, 12 and 24 h of incubation for p38 MAPK and ERK pathways members ( C , D ), the levels of individual proteins were determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments. When assessing cell growth and viability ( A ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). * p
    Figure Legend Snippet: Effect of 1 mM stearic acid (SA) (see “Materials and Methods”) on ( A ) cell growth and viability; ( B ) the level of cleaved PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis); ( C ) the level of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (p38 MAPK signaling pathway); and ( D ) the level of phospho-c-Raf, phospho-MEK1/2, ERK1/2, phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without SA represented control cells. After 18 h of incubation (see “Materials and Methods”) for markers of apoptosis ( B ) and 3, 6, 12 and 24 h of incubation for p38 MAPK and ERK pathways members ( C , D ), the levels of individual proteins were determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments. When assessing cell growth and viability ( A ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). * p

    Techniques Used: Incubation, Western Blot, Concentration Assay

    Effect of the specific p38 MAPK activator, anisomycin, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); ( C ) cell growth and viability; and ( D ) cleavage of PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis) in NES2Y cells. Cells incubated without the activator and SA represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B , D ), the level of individual proteins was determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p
    Figure Legend Snippet: Effect of the specific p38 MAPK activator, anisomycin, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); ( C ) cell growth and viability; and ( D ) cleavage of PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis) in NES2Y cells. Cells incubated without the activator and SA represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B , D ), the level of individual proteins was determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p

    Techniques Used: Incubation, Western Blot, Concentration Assay

    Effect of the specific p38 MAPK inhibitor, SB202190, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without the inhibitor represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of control cells and cells treated with SB202190 as well as when comparing the effect of SA alone and applied together with SB202190.
    Figure Legend Snippet: Effect of the specific p38 MAPK inhibitor, SB202190, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without the inhibitor represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of control cells and cells treated with SB202190 as well as when comparing the effect of SA alone and applied together with SB202190.

    Techniques Used: Incubation, Western Blot, Concentration Assay

    2) Product Images from "p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells"

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17020159

    Effect of p38 MAPK silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.
    Figure Legend Snippet: Effect of p38 MAPK silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.

    Techniques Used: Incubation, Western Blot, Concentration Assay, Negative Control

    Effect of p38 MAPK overexpression, using transfection with a specific plasmid (Vector with p38 MAPK) (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells transfected with an empty vector (Empty vector) represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. The fact that the band of p38 MAPK in the control samples is not visible here resulted from a large difference in p38 MAPK content in control and transfected cells. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with plasmid DNA containing p38 MAPK (Vector with p38 MAPK) and cells incubated with empty plasmid DNA (empty vector).
    Figure Legend Snippet: Effect of p38 MAPK overexpression, using transfection with a specific plasmid (Vector with p38 MAPK) (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells transfected with an empty vector (Empty vector) represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. The fact that the band of p38 MAPK in the control samples is not visible here resulted from a large difference in p38 MAPK content in control and transfected cells. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with plasmid DNA containing p38 MAPK (Vector with p38 MAPK) and cells incubated with empty plasmid DNA (empty vector).

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Incubation, Western Blot, Concentration Assay

    Effect of 1 mM stearic acid (SA) (see “Materials and Methods”) on ( A ) cell growth and viability; ( B ) the level of cleaved PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis); ( C ) the level of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (p38 MAPK signaling pathway); and ( D ) the level of phospho-c-Raf, phospho-MEK1/2, ERK1/2, phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without SA represented control cells. After 18 h of incubation (see “Materials and Methods”) for markers of apoptosis ( B ) and 3, 6, 12 and 24 h of incubation for p38 MAPK and ERK pathways members ( C , D ), the levels of individual proteins were determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments. When assessing cell growth and viability ( A ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). * p
    Figure Legend Snippet: Effect of 1 mM stearic acid (SA) (see “Materials and Methods”) on ( A ) cell growth and viability; ( B ) the level of cleaved PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis); ( C ) the level of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (p38 MAPK signaling pathway); and ( D ) the level of phospho-c-Raf, phospho-MEK1/2, ERK1/2, phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without SA represented control cells. After 18 h of incubation (see “Materials and Methods”) for markers of apoptosis ( B ) and 3, 6, 12 and 24 h of incubation for p38 MAPK and ERK pathways members ( C , D ), the levels of individual proteins were determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments. When assessing cell growth and viability ( A ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). * p

    Techniques Used: Incubation, Western Blot, Concentration Assay

    Effect of the specific p38 MAPK activator, anisomycin, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); ( C ) cell growth and viability; and ( D ) cleavage of PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis) in NES2Y cells. Cells incubated without the activator and SA represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B , D ), the level of individual proteins was determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p
    Figure Legend Snippet: Effect of the specific p38 MAPK activator, anisomycin, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); ( C ) cell growth and viability; and ( D ) cleavage of PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis) in NES2Y cells. Cells incubated without the activator and SA represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B , D ), the level of individual proteins was determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p

    Techniques Used: Incubation, Western Blot, Concentration Assay

    Effect of the specific p38 MAPK inhibitor, SB202190, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without the inhibitor represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of control cells and cells treated with SB202190 as well as when comparing the effect of SA alone and applied together with SB202190.
    Figure Legend Snippet: Effect of the specific p38 MAPK inhibitor, SB202190, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without the inhibitor represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of control cells and cells treated with SB202190 as well as when comparing the effect of SA alone and applied together with SB202190.

    Techniques Used: Incubation, Western Blot, Concentration Assay

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    Expressing:

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    Quantitative RT-PCR:

    Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization
    Article Snippet: The numbers of live cells were determined by hemocytometer counting following trypan blue staining. .. For the prevention study using the p38 MAPK inhibitors, SB203580 or SB202190 (10 μM, Abcam, Cambridge, MA, USA) was applied with the pro-inflammatory cytokines at the beginning of induction and again at day three; expression of endothelial and EndoMT markers was analyzed by RT-qPCR at day six. .. For the intervention study, SB203580 was added at days six, nine and 12 post-induction, and gene expression was analyzed at day 15.

    Concentration Assay:

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells
    Article Snippet: After 48 h of incubation with or without SA, the number of living cells was determined using a hemocytometer counting system after staining with trypan blue. .. Inhibitor and Activator Application Cells (approximately 5 × 105 cells per sample) were seeded and after a 24-h pre-incubation period (allowing cells to attach) the culture medium was replaced with: (1) a serum-free medium with or without the p38 MAPK inhibitor SB202190 (Abcam, Cambridge, UK) at a desired concentration; (2) a serum-free medium containing 2% BSA with or without the p38 MAPK activator anisomycin (Sigma Aldrich, St. Louis, MO, USA) at required concentration; or (3) a serum-free medium containing 2% BSA and SA. ..

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    Abcam p38 mapk inhibitor sb202190
    Effect of <t>p38</t> <t>MAPK</t> silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.
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    Effect of p38 MAPK silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.

    Journal: International Journal of Molecular Sciences

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    doi: 10.3390/ijms17020159

    Figure Lengend Snippet: Effect of p38 MAPK silencing, using a specific siRNA (see “Materials and Methods”) and the effect of stearic acid (SA), on ( A ) the level of p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without siRNA represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Non-specific siRNA was used as a negative control. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with p38 MAPK specific siRNA and with non-specific siRNA.

    Article Snippet: The p38 MAPK inhibitor SB202190, the activator anisomycin, and SA were applied in the same way as described above (“Inhibitor and activator application”).

    Techniques: Incubation, Western Blot, Concentration Assay, Negative Control

    Effect of p38 MAPK overexpression, using transfection with a specific plasmid (Vector with p38 MAPK) (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells transfected with an empty vector (Empty vector) represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. The fact that the band of p38 MAPK in the control samples is not visible here resulted from a large difference in p38 MAPK content in control and transfected cells. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with plasmid DNA containing p38 MAPK (Vector with p38 MAPK) and cells incubated with empty plasmid DNA (empty vector).

    Journal: International Journal of Molecular Sciences

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    doi: 10.3390/ijms17020159

    Figure Lengend Snippet: Effect of p38 MAPK overexpression, using transfection with a specific plasmid (Vector with p38 MAPK) (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells transfected with an empty vector (Empty vector) represented control cells. After 18 h of incubation (see “Materials and Methods”) with or without stearic acid (SA) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. The fact that the band of p38 MAPK in the control samples is not visible here resulted from a large difference in p38 MAPK content in control and transfected cells. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation with or without SA. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of cells incubated with plasmid DNA containing p38 MAPK (Vector with p38 MAPK) and cells incubated with empty plasmid DNA (empty vector).

    Article Snippet: The p38 MAPK inhibitor SB202190, the activator anisomycin, and SA were applied in the same way as described above (“Inhibitor and activator application”).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Incubation, Western Blot, Concentration Assay

    Effect of 1 mM stearic acid (SA) (see “Materials and Methods”) on ( A ) cell growth and viability; ( B ) the level of cleaved PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis); ( C ) the level of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (p38 MAPK signaling pathway); and ( D ) the level of phospho-c-Raf, phospho-MEK1/2, ERK1/2, phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without SA represented control cells. After 18 h of incubation (see “Materials and Methods”) for markers of apoptosis ( B ) and 3, 6, 12 and 24 h of incubation for p38 MAPK and ERK pathways members ( C , D ), the levels of individual proteins were determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments. When assessing cell growth and viability ( A ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). * p

    Journal: International Journal of Molecular Sciences

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    doi: 10.3390/ijms17020159

    Figure Lengend Snippet: Effect of 1 mM stearic acid (SA) (see “Materials and Methods”) on ( A ) cell growth and viability; ( B ) the level of cleaved PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis); ( C ) the level of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, phospho-MAPKAPK-2 (p38 MAPK signaling pathway); and ( D ) the level of phospho-c-Raf, phospho-MEK1/2, ERK1/2, phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without SA represented control cells. After 18 h of incubation (see “Materials and Methods”) for markers of apoptosis ( B ) and 3, 6, 12 and 24 h of incubation for p38 MAPK and ERK pathways members ( C , D ), the levels of individual proteins were determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments. When assessing cell growth and viability ( A ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). * p

    Article Snippet: The p38 MAPK inhibitor SB202190, the activator anisomycin, and SA were applied in the same way as described above (“Inhibitor and activator application”).

    Techniques: Incubation, Western Blot, Concentration Assay

    Effect of the specific p38 MAPK activator, anisomycin, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); ( C ) cell growth and viability; and ( D ) cleavage of PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis) in NES2Y cells. Cells incubated without the activator and SA represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B , D ), the level of individual proteins was determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    doi: 10.3390/ijms17020159

    Figure Lengend Snippet: Effect of the specific p38 MAPK activator, anisomycin, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); ( C ) cell growth and viability; and ( D ) cleavage of PARP, caspase-7 (C7), caspase-8 (C8) and caspase-9 (C9) (markers of apoptosis) in NES2Y cells. Cells incubated without the activator and SA represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B , D ), the level of individual proteins was determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p

    Article Snippet: The p38 MAPK inhibitor SB202190, the activator anisomycin, and SA were applied in the same way as described above (“Inhibitor and activator application”).

    Techniques: Incubation, Western Blot, Concentration Assay

    Effect of the specific p38 MAPK inhibitor, SB202190, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without the inhibitor represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of control cells and cells treated with SB202190 as well as when comparing the effect of SA alone and applied together with SB202190.

    Journal: International Journal of Molecular Sciences

    Article Title: p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    doi: 10.3390/ijms17020159

    Figure Lengend Snippet: Effect of the specific p38 MAPK inhibitor, SB202190, (see “Materials and Methods”) and the effect of stearic acid (SA) on ( A ) the level of phospho-MAPKAPK-2 (substrate of p38 MAPK); ( B ) the level of phospho-c-Raf, phospho-MEK1/2, phospho-ERK1/2 (the ERK signaling pathway); and ( C ) cell growth and viability of NES2Y cells. Cells incubated without the inhibitor represented control cells. After 12 h of incubation (see “Materials and Methods”) ( A , B ), the level of individual proteins was determined using Western blot analysis and the relevant antibodies (see “Materials and Methods”). A monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from three independent experiments. When assessing cell growth and viability ( C ), cells were seeded at a concentration of 2 × 10 4 cells/100 µL of culture medium per well of 96-well plate (see “Materials and Methods”). The number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. NS (non-significant) when comparing the number of control cells and cells treated with SB202190 as well as when comparing the effect of SA alone and applied together with SB202190.

    Article Snippet: The p38 MAPK inhibitor SB202190, the activator anisomycin, and SA were applied in the same way as described above (“Inhibitor and activator application”).

    Techniques: Incubation, Western Blot, Concentration Assay