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732a rrid ab 2237618 (Bethyl)

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Bethyl 732a rrid ab 2237618
732a Rrid Ab 2237618, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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732a rrid ab 2237618 - by Bioz Stars, 2026-03

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(A) PPP2R1A overexpression promotes RTA dephosphorylation. iSLK.RGB-Vector and iSLK.RGB -PPP2R1A cells were induced with doxycycline at different time points as indicated. Cells were then lysed, and cell lysates were immunoprecipitated with an anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (B) PP2A activity was measured among iSLK.RGB-Vector and iSLK.RGB-PPP2R1A cells. Bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). ****P < 0.0001. (C) Phosphatase PP2A agonist Forskolin promotes RTA dephosphorylation. iSLK.RGB cells were induced with doxycycline for 48 hours in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (D) Phosphatase PP2A inhibitor LB-100 enhances RTA phosphorylation. iSLK.RGB cells were induced with doxycycline for 48 hours in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (E) Phosphatase PP2A agonist Forskolin cannot promote RTA dephosphorylation when PPP2R1A expression was suppressed with siRNAs. iSLK.RGB cells were transfected with siRNA as indicated. At 24 hours after transfection, cells were induced by doxycycline for 48 hours in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level.

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: (A) PPP2R1A overexpression promotes RTA dephosphorylation. iSLK.RGB-Vector and iSLK.RGB -PPP2R1A cells were induced with doxycycline at different time points as indicated. Cells were then lysed, and cell lysates were immunoprecipitated with an anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (B) PP2A activity was measured among iSLK.RGB-Vector and iSLK.RGB-PPP2R1A cells. Bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). ****P < 0.0001. (C) Phosphatase PP2A agonist Forskolin promotes RTA dephosphorylation. iSLK.RGB cells were induced with doxycycline for 48 hours in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (D) Phosphatase PP2A inhibitor LB-100 enhances RTA phosphorylation. iSLK.RGB cells were induced with doxycycline for 48 hours in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (E) Phosphatase PP2A agonist Forskolin cannot promote RTA dephosphorylation when PPP2R1A expression was suppressed with siRNAs. iSLK.RGB cells were transfected with siRNA as indicated. At 24 hours after transfection, cells were induced by doxycycline for 48 hours in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity gel (Sigma, A2220), Lipofectamine 2000 (ThermoFisher Scientific, 11668019), MG132 (MedChemExpress, HY-13259), cycloheximide (CHX) (MedChemExpress, HY-12320), protease inhibitor cocktail (Sigma, P8340), phosphatase PP2A inhibitor (LB-100, MedChemExpress, HY-18597) and phosphatase PP2A agonist (Forskolin, MedChemExpress, HY-15371).

Techniques: Over Expression, De-Phosphorylation Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, Activity Assay, Phospho-proteomics, Expressing, Transfection

(A) Phosphatase PP2A agonist Forskolin suppresses the transcription of viral genes. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). RNA was extracted from cells to investigate the transcriptional level of several KSHV genes: ORF71, ORF45, ORF57 and K8.1. (B) Phosphatase PP2A inhibitor LB-100 promotes the transcription of viral genes. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). RNA was extracted from cells to investigate the transcriptional level of several KSHV genes: ORF71, ORF45, ORF57 and K8.1. (C) Phosphatase PP2A agonist Forskolin suppresses virus production. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). Extracellular virion DNA were extracted from cell supernatants. Then, the KSHV genomic DNA copy numbers were quantified by qPCR analysis. (D) Phosphatase PP2A inhibitor LB-100 promotes virus production. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). Extracellular virion DNA were extracted from cell supernatants. Then, the KSHV genomic DNA copy numbers were quantified by qPCR analysis. For A to D, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: (A) Phosphatase PP2A agonist Forskolin suppresses the transcription of viral genes. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). RNA was extracted from cells to investigate the transcriptional level of several KSHV genes: ORF71, ORF45, ORF57 and K8.1. (B) Phosphatase PP2A inhibitor LB-100 promotes the transcription of viral genes. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). RNA was extracted from cells to investigate the transcriptional level of several KSHV genes: ORF71, ORF45, ORF57 and K8.1. (C) Phosphatase PP2A agonist Forskolin suppresses virus production. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). Extracellular virion DNA were extracted from cell supernatants. Then, the KSHV genomic DNA copy numbers were quantified by qPCR analysis. (D) Phosphatase PP2A inhibitor LB-100 promotes virus production. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). Extracellular virion DNA were extracted from cell supernatants. Then, the KSHV genomic DNA copy numbers were quantified by qPCR analysis. For A to D, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Techniques: Virus

(A) Phosphatase PP2A agonist Forskolin suppresses the transcription activity of RTA. (B) Phosphatase PP2A inhibitor LB-100 promotes the transcription activity of RTA. For A and B, HEK293T cells were transfected with ORF57 (left) or PAN (right) reporter plasmids (1 μg) and expression plasmids containing RTA (1 μg) or empty vector (1 μg) as a control. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (A) or 5 μM PP2A inhibitor LB-100 (B) for 48 hours. Cells were then lysed to detect dual luciferase reporter activity and cell lysates were immunoprecipitated with an anti-Flag antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (C) PP2A enzymatic activity was measured in two peptides of RTA containing phosphorylated Thr-42 or Thr-678 sites respectively and one random peptide. (D and E) Phosphatase PP2A agonist Forskolin (D) or inhibitor LB-100 (E) has no effect on the phosphorylation status of RTA mutants containing T42A and T678A sites. For D and E, HEK293T cells were transfected with wildtype RTA or RTA mutants as indicated. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (D) or 5 μM PP2A inhibitor LB-100 (E) for 48 hours. Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (F) The transcriptional activity of three RTA mutants was impaired. HEK293T cells were transfected with K8 (left) or ORF59 (right) reporter plasmids (1 μg) and expression plasmids containing wildtype RTA or RTA mutants as indicated (1 μg) or empty vector (1 μg) as a control. At 48 hours after transfection, cells were then lysed to detect dual luciferase reporter activity. (G and H) Phosphatase PP2A agonist Forskolin (G) or inhibitor LB-100 (H) has no effect on the transcriptional activity of RTA mutants containing T42A and T678A sites. For G and H, HEK293T cells were transfected with K8 (left panel) or ORF59 (right) reporter plasmids (1 μg) and expression plasmids containing wildtype RTA or RTA mutants as indicated (1 μg) or empty vector (1 μg) as a control. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (G) or 5 μM PP2A inhibitor LB-100 (H) for 48 hours. Cells were then lysed to detect dual luciferase reporter activity. For A to C and F to H, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates not significant.

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: (A) Phosphatase PP2A agonist Forskolin suppresses the transcription activity of RTA. (B) Phosphatase PP2A inhibitor LB-100 promotes the transcription activity of RTA. For A and B, HEK293T cells were transfected with ORF57 (left) or PAN (right) reporter plasmids (1 μg) and expression plasmids containing RTA (1 μg) or empty vector (1 μg) as a control. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (A) or 5 μM PP2A inhibitor LB-100 (B) for 48 hours. Cells were then lysed to detect dual luciferase reporter activity and cell lysates were immunoprecipitated with an anti-Flag antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (C) PP2A enzymatic activity was measured in two peptides of RTA containing phosphorylated Thr-42 or Thr-678 sites respectively and one random peptide. (D and E) Phosphatase PP2A agonist Forskolin (D) or inhibitor LB-100 (E) has no effect on the phosphorylation status of RTA mutants containing T42A and T678A sites. For D and E, HEK293T cells were transfected with wildtype RTA or RTA mutants as indicated. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (D) or 5 μM PP2A inhibitor LB-100 (E) for 48 hours. Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (F) The transcriptional activity of three RTA mutants was impaired. HEK293T cells were transfected with K8 (left) or ORF59 (right) reporter plasmids (1 μg) and expression plasmids containing wildtype RTA or RTA mutants as indicated (1 μg) or empty vector (1 μg) as a control. At 48 hours after transfection, cells were then lysed to detect dual luciferase reporter activity. (G and H) Phosphatase PP2A agonist Forskolin (G) or inhibitor LB-100 (H) has no effect on the transcriptional activity of RTA mutants containing T42A and T678A sites. For G and H, HEK293T cells were transfected with K8 (left panel) or ORF59 (right) reporter plasmids (1 μg) and expression plasmids containing wildtype RTA or RTA mutants as indicated (1 μg) or empty vector (1 μg) as a control. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (G) or 5 μM PP2A inhibitor LB-100 (H) for 48 hours. Cells were then lysed to detect dual luciferase reporter activity. For A to C and F to H, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates not significant.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Immunoprecipitation, Western Blot, Phospho-proteomics

The scaffold protein PPP2R1A interacted with RTA, inducing RTA dephosphorylation mediated by phosphatase PP2A, which seriously destroyed the transcription activity of RTA and thereby greatly inhibiting KSHV lytic replication. In turn, RTA promoted PPP2R1A degradation through ubiquitin-proteasome pathway, counteracting the antiviral activity of phosphatase PP2A and ensuring a complete lytic replication of KSHV.

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: The scaffold protein PPP2R1A interacted with RTA, inducing RTA dephosphorylation mediated by phosphatase PP2A, which seriously destroyed the transcription activity of RTA and thereby greatly inhibiting KSHV lytic replication. In turn, RTA promoted PPP2R1A degradation through ubiquitin-proteasome pathway, counteracting the antiviral activity of phosphatase PP2A and ensuring a complete lytic replication of KSHV.

Techniques: De-Phosphorylation Assay, Activity Assay, Ubiquitin Proteomics

a Experimental scheme to test factors that regulate polar chromosome congression following washout of a CENP-E inhibitor. b Percentage of polar chromosomes per cell that successfully aligned within 30 min of washout under the indicated treatments. Colored points represent individual cells; black lines show the mean, with light and dark gray areas marking 95% confidence intervals for the mean and standard deviation, respectively. Numbers: 23, 4, 13, 24, 11, 23, 21, 18, 10 cells, all from ≥3 independent biological replicates. Representative images of CENP-A-GFP and centrin1-GFP expressing RPE-1 cells (centrioles circled) over time after washout of CENP-E inhibitor GSK-923295 in phosphatase-pre-inhibited (Okadaic acid) ( c ), Aurora B acutely inhibited (ZM447439) ( d ), Spindly-depleted ( e ) and BuBR1-depleted ( f ) cells. Images are maximum projections color-coded by depth (color bar in Fig. ). Arrowheads mark polar kinetochores failing to congress. Statistics: two-tailed ANOVA with Tukey’s HSD post hoc test. Symbols: ****, P ≤ 0.0001; inh., inhibited; depl., depleted; siRNA, small interfering RNA; PP, protein phosphatases; KK, Kid and Kif4a; chrom., chromosome. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on attachments

doi: 10.1038/s41467-025-64148-w

Figure Lengend Snippet: a Experimental scheme to test factors that regulate polar chromosome congression following washout of a CENP-E inhibitor. b Percentage of polar chromosomes per cell that successfully aligned within 30 min of washout under the indicated treatments. Colored points represent individual cells; black lines show the mean, with light and dark gray areas marking 95% confidence intervals for the mean and standard deviation, respectively. Numbers: 23, 4, 13, 24, 11, 23, 21, 18, 10 cells, all from ≥3 independent biological replicates. Representative images of CENP-A-GFP and centrin1-GFP expressing RPE-1 cells (centrioles circled) over time after washout of CENP-E inhibitor GSK-923295 in phosphatase-pre-inhibited (Okadaic acid) ( c ), Aurora B acutely inhibited (ZM447439) ( d ), Spindly-depleted ( e ) and BuBR1-depleted ( f ) cells. Images are maximum projections color-coded by depth (color bar in Fig. ). Arrowheads mark polar kinetochores failing to congress. Statistics: two-tailed ANOVA with Tukey’s HSD post hoc test. Symbols: ****, P ≤ 0.0001; inh., inhibited; depl., depleted; siRNA, small interfering RNA; PP, protein phosphatases; KK, Kid and Kif4a; chrom., chromosome. Source data are provided as a Source Data file.

Article Snippet: Inhibitor of PP1 and PP2A Okadaic acid (MedChemExpress, IC 50 value 0.1–0.3 nM for PP2A and 15–50 nM for PP1) at a final concentration of 1 μM, was added acutely before imaging or 30 min before washout of the CENP-E inhibitor as noted.

Techniques: Standard Deviation, Expressing, Two Tailed Test, Small Interfering RNA

a Aurora kinases inhibit congression initiation by phosphorylating the Ndc80 tail near centrosomes (1). On polar kinetochores, CENP-E–BubR1 facilitates early end-on attachment formation near the Aurora A gradient (2), triggering a decline in Aurora B activity, loss of Mad2 from the kinetochore, and stabilization of Ndc80-microtubule binding, all preceding fast kinetochore movement (3). b This establishes a negative feedback loop involving Aurora B, CENP-E, BubR1-PP2A, the fibrous corona, and outer kinetochore proteins (Knl1 and Hec1/Ndc80). The loop self-limits by promoting end-on attachment and initiating chromosome congression, which in turn reduces the upstream signals that sustain it. Lines with arrows indicate activation, and blunt lines indicate deactivation by phosphorylation or direct physical inhibition. Initially, polar chromosomes form lateral kinetochore-microtubule attachments that fail to convert to stable end-on attachments without CENP-E due to high Aurora B activity. Aurora B phosphorylates Knl1 and Hec1 to prevent end-on conversion and maintains the expanded fibrous corona (depicted as a crown), which further inhibits attachment stabilization. Aurora B also activates CENP-E via phosphorylation. We propose (indicated by a question mark) that activated CENP-E interacts with BubR1 and possibly PP2A to counteract Aurora B-mediated phosphorylation, enabling stabilization of initial end-on attachments, progressive corona disassembly, and chromosome congression toward the spindle midplane.

Journal: Nature Communications

Article Title: CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on attachments

doi: 10.1038/s41467-025-64148-w

Figure Lengend Snippet: a Aurora kinases inhibit congression initiation by phosphorylating the Ndc80 tail near centrosomes (1). On polar kinetochores, CENP-E–BubR1 facilitates early end-on attachment formation near the Aurora A gradient (2), triggering a decline in Aurora B activity, loss of Mad2 from the kinetochore, and stabilization of Ndc80-microtubule binding, all preceding fast kinetochore movement (3). b This establishes a negative feedback loop involving Aurora B, CENP-E, BubR1-PP2A, the fibrous corona, and outer kinetochore proteins (Knl1 and Hec1/Ndc80). The loop self-limits by promoting end-on attachment and initiating chromosome congression, which in turn reduces the upstream signals that sustain it. Lines with arrows indicate activation, and blunt lines indicate deactivation by phosphorylation or direct physical inhibition. Initially, polar chromosomes form lateral kinetochore-microtubule attachments that fail to convert to stable end-on attachments without CENP-E due to high Aurora B activity. Aurora B phosphorylates Knl1 and Hec1 to prevent end-on conversion and maintains the expanded fibrous corona (depicted as a crown), which further inhibits attachment stabilization. Aurora B also activates CENP-E via phosphorylation. We propose (indicated by a question mark) that activated CENP-E interacts with BubR1 and possibly PP2A to counteract Aurora B-mediated phosphorylation, enabling stabilization of initial end-on attachments, progressive corona disassembly, and chromosome congression toward the spindle midplane.

Techniques: Activity Assay, Binding Assay, Activation Assay, Phospho-proteomics, Inhibition

MEK/ERK signaling inhibition reduces glioblastoma-induced neuronal ERK activation and spine loss. (A) Representative immunofluorescence images of total ERK1/2 (green) and phosphorylated ERK1/2 (p-ERK1/2, red) in neurons under different conditions: control (Ctrl-neuron), exposed to U-87 MG glioblastoma cells (U-87 MG-neuron), U-87 MG-exposed neurons treated with an ERK peptide inhibitor (U-87 MG-neuron +ERK inhibitor), or U-87 MG-exposed neurons treated with the MEK inhibitor Selumetinib (U-87 MG-neuron +MEK inhibitor). Nuclei were stained with DAPI (blue). Scale bars = 30 µm. (B) Fluorescent phalloidin staining (gray) of dendritic spines in neurons after 35 days of differentiation under the same conditions as in (A). Insets (b1-b4) show magnified views of the dashed regions. Scale bars = 30 µm. (C-D) Quantification of immunofluorescence intensity of total ERK1/2 (C) and p-ERK1/2 (D) in neurons under each condition from (B). Data are pooled from 21-67 randomly selected neuronal fields per independent experiment. Values represent mean ± SD. Statistical comparisons were performed using unpaired t-tests. Significance: ns (not significant), ***P < 0.001, ****P < 0.0001. (E) Quantification of dendritic spine density (spines per 10 µm) under each condition from (B). Data represent 72-160 dendritic segments from 2-3 independent experiments. Values are shown as mean ± SD. Unpaired t-tests were used for statistical analysis; ****P < 0.0001. (F-G) Phase-contrast images (F) and quantification (G) of HMC3 microglia and U-87 MG cells imaged using Incucyte. Treatment with Selumetinib (MEK inhibitor) or Selumetinib combined with DT-061 (MEK/PP2A pathway inhibition) reduced U-87 MG cell proliferation and migration, and induced cell death. Scale bars = 30 µm. Values are shown as mean ± SEM. Data are pooled from 4 cultures per each condition; 5 fields were imaged per each culture.

Journal: bioRxiv

Article Title: A Human Neuronal Co-Culture System Reveals Early Tumor-Neuron Communication and Targetable Pathways in Glioblastoma

doi: 10.1101/2025.09.10.675396

Figure Lengend Snippet: MEK/ERK signaling inhibition reduces glioblastoma-induced neuronal ERK activation and spine loss. (A) Representative immunofluorescence images of total ERK1/2 (green) and phosphorylated ERK1/2 (p-ERK1/2, red) in neurons under different conditions: control (Ctrl-neuron), exposed to U-87 MG glioblastoma cells (U-87 MG-neuron), U-87 MG-exposed neurons treated with an ERK peptide inhibitor (U-87 MG-neuron +ERK inhibitor), or U-87 MG-exposed neurons treated with the MEK inhibitor Selumetinib (U-87 MG-neuron +MEK inhibitor). Nuclei were stained with DAPI (blue). Scale bars = 30 µm. (B) Fluorescent phalloidin staining (gray) of dendritic spines in neurons after 35 days of differentiation under the same conditions as in (A). Insets (b1-b4) show magnified views of the dashed regions. Scale bars = 30 µm. (C-D) Quantification of immunofluorescence intensity of total ERK1/2 (C) and p-ERK1/2 (D) in neurons under each condition from (B). Data are pooled from 21-67 randomly selected neuronal fields per independent experiment. Values represent mean ± SD. Statistical comparisons were performed using unpaired t-tests. Significance: ns (not significant), ***P < 0.001, ****P < 0.0001. (E) Quantification of dendritic spine density (spines per 10 µm) under each condition from (B). Data represent 72-160 dendritic segments from 2-3 independent experiments. Values are shown as mean ± SD. Unpaired t-tests were used for statistical analysis; ****P < 0.0001. (F-G) Phase-contrast images (F) and quantification (G) of HMC3 microglia and U-87 MG cells imaged using Incucyte. Treatment with Selumetinib (MEK inhibitor) or Selumetinib combined with DT-061 (MEK/PP2A pathway inhibition) reduced U-87 MG cell proliferation and migration, and induced cell death. Scale bars = 30 µm. Values are shown as mean ± SEM. Data are pooled from 4 cultures per each condition; 5 fields were imaged per each culture.

Article Snippet: In inhibition experiments, cells were seed as above and treated twenty-four hours after with varying concentrations of a MEK inhibitor (selumetinib, MedChemExpress, Cat. # HY-50706), ERK inhibitor peptide [ ], or a PP2A activator (DT-061, MedChemExpress, Cat. # HY-112929).

Techniques: Inhibition, Activation Assay, Immunofluorescence, Control, Staining, Migration

Lactotransferrin (LTF) competitively bound p65. A , Phosphorylation levels of p65 in control or LTF-depleted glioblastoma (GBM) cells was analyzed by western blot. B , U251 cells were transfected with LTF with or without four phosphatases. Phosphorylation levels of p65 in control or LTF-depleted GBM cells were analyzed by western blot. C , Immunoprecipitates were prepared with anti-LTF antibody. p65 and PP2A expression in immunoprecipitates was measured by western blot. D , Control and LTF-depleted cells were transfected with Flag-p65. Immunoprecipitates were prepared with anti-Flag antibody. PP2A expression in immunoprecipitates was measured by western blot. E , Schematic representation of Flag-tagged full-length LTF (#1) and various deletion mutants (#2-4). F , Immunoprecipitation and western blot assays using Flag antibody showing the interaction between p65 and LTF protein in U251 cells transfected with a series of truncations of Flag-tagged LTF. G , Schematic representation of Flag-tagged full-length p65 (#1) and various deletion mutants (#2-5). H , Immunoprecipitation and western blot assays using Flag antibody showing the interaction between p65 and LTF protein in U251 cells transfected with a series truncations of Flag-tagged p65.

Journal: Brazilian Journal of Medical and Biological Research

Article Title: LTF regulates glioblastoma progression and temozolomide resistance via the NF-κB signaling pathway

doi: 10.1590/1414-431X2025e13821

Figure Lengend Snippet: Lactotransferrin (LTF) competitively bound p65. A , Phosphorylation levels of p65 in control or LTF-depleted glioblastoma (GBM) cells was analyzed by western blot. B , U251 cells were transfected with LTF with or without four phosphatases. Phosphorylation levels of p65 in control or LTF-depleted GBM cells were analyzed by western blot. C , Immunoprecipitates were prepared with anti-LTF antibody. p65 and PP2A expression in immunoprecipitates was measured by western blot. D , Control and LTF-depleted cells were transfected with Flag-p65. Immunoprecipitates were prepared with anti-Flag antibody. PP2A expression in immunoprecipitates was measured by western blot. E , Schematic representation of Flag-tagged full-length LTF (#1) and various deletion mutants (#2-4). F , Immunoprecipitation and western blot assays using Flag antibody showing the interaction between p65 and LTF protein in U251 cells transfected with a series of truncations of Flag-tagged LTF. G , Schematic representation of Flag-tagged full-length p65 (#1) and various deletion mutants (#2-5). H , Immunoprecipitation and western blot assays using Flag antibody showing the interaction between p65 and LTF protein in U251 cells transfected with a series truncations of Flag-tagged p65.

Article Snippet: Antibodies against LTF (10933-1-AP), Cyclin E1 (11554-1-AP), Cyclin D1 (26939-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), γ-H2AX (29380-1-AP), PP2A (13482-1-AP), GAPDH (10494-1-AP), and H3 (17168-1-AP) were from Proteintech (China).

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

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