plcb1  (Santa Cruz Biotechnology)

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: A NT2 cells progenitors were treated with RA and immunoblotted with anti-PLCβ1a and PLCβ1b antibodies at various times (48 h, 7 d, 14 d, 28 d). Terminally differentiated NT2N cells were isolated from cultures at 28 d of RA exposue by mechanical dislogding (see Methods). Equal amounts of total protein (12 μg) were loaded ( n = 4). B Similar studies as in ( A ) except NT2 progenitors were treated with AraC at various times (12, 24, 48 and 72 h and 6 days -NT2N cells-) where time points were chosen from previous studies . Equal amounts of total protein (12 μg) were loaded. The optical density (OD) is expressed as the percentage of immunoreactive signal found in NT2 progenitor cells (100%). Repeated measures one-way ANOVA followed by Tukey’s post hoc test. Significant differences between the different samples with respect to NT2 progenitors are shown (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are mean ± SEM ( n = 4). C Immunoblots of PLCβ1 on whole homogenates of NT2 progenitors, AraC-treated cells, and AraC/NT2N cells and NF200 and βIII-tubulin. Equal amounts of total protein (12 μg) were loaded.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Isolation, Western Blot

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: Immunofluorescence images using anti-PLCβ1 (red), NF200 (green) and Hoechst’s chromatin staining (blue) of NT2 progenitors ( A ) and after treatment with RA ( A – F ) or AraC ( G – I ). Cells treated with RA were fixed and immunostained at 48 h ( B ), 7 days ( C ), 28 days ( D ), and after isolation of terminally differentiated NT2N neurons by mechanical dislodging ( E ). Non-neuronal cells (NT2A) that remained attached to the culture flask after the mechanical isolation of NT2N neurons were also doubly immunostained for PLCβ1 and NF200 ( F ). Cells treated with AraC were fixed at 48 h ( G ), 72 h ( H ) and 6 days, when cells were differentiated into NT2N neurons ( I ). Images are maximum intensity projections of four consecutive optical sections separated by 0.24 µm and obtained using a structured illumination module. Scale bar in F = 20 µm (applies to A – F ). Scale bar in I = 20 µm (applies to G – I ).

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Immunofluorescence, Staining, Isolation

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: Sample Western blot ( A ) and densitometric analysis ( B ) of PLCβ1, NF200 and βIII-tubulin expression in NT2 cells transfected either with scrambled off-target siRNA or siRNA targeting the PLCB1 transcript (PLCβ1 siRNA) and treated with AraC for 72 h. Equal amounts of total protein (30 μg) were loaded on the same gel and run in parallel. The y -axis of the graph shows the mean optical density (OD) as a percentage of that in NT2 cells (100%). One-way ANOVA followed by post hoc Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are mean ± SEM ( n = 4). C – D Immunofluorescence visualizing PLCβ1 (red) and βIII-tubulin (green) combined with Hoechst’s chromatin staining (blue) in NT2 cells after transfection with either scrambled siRNA ( C ) or PLCβ1 siRNA ( D ) and treated with AraC for 72 h. Scale bar = 100 µm (applies to C , D ). E–N Immunofluorescence studies of primary rat astrocytes under control conditions or treated with si(RNA)PLCβ1a where NF-H refers to the heavy subunit of neurofilament. More images can be found in Fig. S . Scale bar = 50 μm.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Staining, Control

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: A – K Studies of NT2 cells. A – B Representative immunoblots ( A ) and densitometric analysis ( B ) for NF200 and βIII-tubulin in homogenates from NT2 cells transfected with either empty pcDNA, pcDNA-PLCβ1a or pcDNA-PLCβ1b and treated with AraC for 96 h. Equal amounts of total protein (5 μg) were loaded. The y -axis in B shows the mean optical density (OD) as a percentage of that in NT2 cells (100%). Significance was assessed by one-way ANOVA followed by post hoc Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are mean ± SEM ( n = 4). C – K are immunofluorescence images of NT2 progenitor cells transfected with empty pCDNA ( C – E ), pcDNA-PLCβ1a ( F – H ) or pcDNA-PLCβ1b ( I – K ) fixed 96 h after transfection and immunostained with anti-PLCβ1 (red) and anti-NF200 (green) antibodies combined with Hoechst’s chromatin staining (blue). Micrographs are maximum intensity projections of four consecutive optical sections, separated by 0.24 µm, obtained using a structured illumination module. Scale bar = 25 µm (applies to C – K ). L – P Results showing that β over-expression of PLCβ1 induces PC12 cell differentiated morphology, where L shows DIC images of mock transfected cells, M cells 24 h after NGF treatment, N cells transfected with PLCβ1a for 24 h and subsequently treated with NGF for 24 h, and O cells over-expressing PLCβ1. Scale bar = 50 μm (applies to L – O ). P Box plots compiling changes in neurite length, where n = 20–41 from 4 independent experiments and significance was assessed by unpaired t test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Western Blot, Transfection, Immunofluorescence, Staining, Over Expression, Expressing

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: A Changes in the nuclear versus total cell intensity of Egr-1 after transfection of NT2 cells with empty plasmid or different PLCβ1 constructs. Data are mean ± SEM ( n = 33). Statistical significance was assessed by one-way ANOVA followed by post hoc Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). B – C Box plots and representative immunofluorescence images showing Egr-1 nuclear levels in PC12 cells exposed to different treatments, including transfection medium alone, complete medium alone and transfection medium combined with complete medium. Plots show min to max vaules as well as individual data points. Significance was assessed by unpaired t test: ns ( P > 0.05), * ( P ≤ 0.05), ** ( P ≤ 0.01), *** ( P ≤ 0.001), and **** ( P ≤ 0.0001). Scale bar in C = 50 µm. D Data showing that upregulation of PLCβ1 in PC12 cells causes the dissociation between TRBP and Egr-1 where TRBP was immunoprecipitated from PC12 cell lysates and Egr-1 levels were quantified by Western blotting. Bar graph shows mean values ± SD as well as individual data points. Unpaired t test was used to assess statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). E Immunofluorescence images probing changes in TRBP and Egr-1 in control HEK293 cells and cells induced to over-express PLCβ1. Scale bar in E = 50 µm.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Transfection, Plasmid Preparation, Construct, Immunofluorescence, Immunoprecipitation, Western Blot, Control

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: At low PLCβ1 levels, Ago2 forms complexes that include TRBP [ , ] which binds Egr-1 in the cytosol [ , ] stabilizing the cytosolic localization of Egr-1. Increasing PLCβ1 helps solubilize Ago2 complexes releasing TRBP promoting nuclear localization of Egr-1, and potentially TRBP.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques:

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: A NT2 cells progenitors were treated with RA and immunoblotted with anti-PLCβ1a and PLCβ1b antibodies at various times (48 h, 7 d, 14 d, 28 d). Terminally differentiated NT2N cells were isolated from cultures at 28 d of RA exposue by mechanical dislogding (see Methods). Equal amounts of total protein (12 μg) were loaded ( n = 4). B Similar studies as in ( A ) except NT2 progenitors were treated with AraC at various times (12, 24, 48 and 72 h and 6 days -NT2N cells-) where time points were chosen from previous studies . Equal amounts of total protein (12 μg) were loaded. The optical density (OD) is expressed as the percentage of immunoreactive signal found in NT2 progenitor cells (100%). Repeated measures one-way ANOVA followed by Tukey’s post hoc test. Significant differences between the different samples with respect to NT2 progenitors are shown (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are mean ± SEM ( n = 4). C Immunoblots of PLCβ1 on whole homogenates of NT2 progenitors, AraC-treated cells, and AraC/NT2N cells and NF200 and βIII-tubulin. Equal amounts of total protein (12 μg) were loaded.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Isolation, Western Blot

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: Immunofluorescence images using anti-PLCβ1 (red), NF200 (green) and Hoechst’s chromatin staining (blue) of NT2 progenitors ( A ) and after treatment with RA ( A – F ) or AraC ( G – I ). Cells treated with RA were fixed and immunostained at 48 h ( B ), 7 days ( C ), 28 days ( D ), and after isolation of terminally differentiated NT2N neurons by mechanical dislodging ( E ). Non-neuronal cells (NT2A) that remained attached to the culture flask after the mechanical isolation of NT2N neurons were also doubly immunostained for PLCβ1 and NF200 ( F ). Cells treated with AraC were fixed at 48 h ( G ), 72 h ( H ) and 6 days, when cells were differentiated into NT2N neurons ( I ). Images are maximum intensity projections of four consecutive optical sections separated by 0.24 µm and obtained using a structured illumination module. Scale bar in F = 20 µm (applies to A – F ). Scale bar in I = 20 µm (applies to G – I ).

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Immunofluorescence, Staining, Isolation

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: Sample Western blot ( A ) and densitometric analysis ( B ) of PLCβ1, NF200 and βIII-tubulin expression in NT2 cells transfected either with scrambled off-target siRNA or siRNA targeting the PLCB1 transcript (PLCβ1 siRNA) and treated with AraC for 72 h. Equal amounts of total protein (30 μg) were loaded on the same gel and run in parallel. The y -axis of the graph shows the mean optical density (OD) as a percentage of that in NT2 cells (100%). One-way ANOVA followed by post hoc Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are mean ± SEM ( n = 4). C – D Immunofluorescence visualizing PLCβ1 (red) and βIII-tubulin (green) combined with Hoechst’s chromatin staining (blue) in NT2 cells after transfection with either scrambled siRNA ( C ) or PLCβ1 siRNA ( D ) and treated with AraC for 72 h. Scale bar = 100 µm (applies to C , D ). E–N Immunofluorescence studies of primary rat astrocytes under control conditions or treated with si(RNA)PLCβ1a where NF-H refers to the heavy subunit of neurofilament. More images can be found in Fig. S . Scale bar = 50 μm.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Staining, Control

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: A – K Studies of NT2 cells. A – B Representative immunoblots ( A ) and densitometric analysis ( B ) for NF200 and βIII-tubulin in homogenates from NT2 cells transfected with either empty pcDNA, pcDNA-PLCβ1a or pcDNA-PLCβ1b and treated with AraC for 96 h. Equal amounts of total protein (5 μg) were loaded. The y -axis in B shows the mean optical density (OD) as a percentage of that in NT2 cells (100%). Significance was assessed by one-way ANOVA followed by post hoc Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are mean ± SEM ( n = 4). C – K are immunofluorescence images of NT2 progenitor cells transfected with empty pCDNA ( C – E ), pcDNA-PLCβ1a ( F – H ) or pcDNA-PLCβ1b ( I – K ) fixed 96 h after transfection and immunostained with anti-PLCβ1 (red) and anti-NF200 (green) antibodies combined with Hoechst’s chromatin staining (blue). Micrographs are maximum intensity projections of four consecutive optical sections, separated by 0.24 µm, obtained using a structured illumination module. Scale bar = 25 µm (applies to C – K ). L – P Results showing that β over-expression of PLCβ1 induces PC12 cell differentiated morphology, where L shows DIC images of mock transfected cells, M cells 24 h after NGF treatment, N cells transfected with PLCβ1a for 24 h and subsequently treated with NGF for 24 h, and O cells over-expressing PLCβ1. Scale bar = 50 μm (applies to L – O ). P Box plots compiling changes in neurite length, where n = 20–41 from 4 independent experiments and significance was assessed by unpaired t test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Western Blot, Transfection, Immunofluorescence, Staining, Over Expression, Expressing

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: A Changes in the nuclear versus total cell intensity of Egr-1 after transfection of NT2 cells with empty plasmid or different PLCβ1 constructs. Data are mean ± SEM ( n = 33). Statistical significance was assessed by one-way ANOVA followed by post hoc Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). B – C Box plots and representative immunofluorescence images showing Egr-1 nuclear levels in PC12 cells exposed to different treatments, including transfection medium alone, complete medium alone and transfection medium combined with complete medium. Plots show min to max vaules as well as individual data points. Significance was assessed by unpaired t test: ns ( P > 0.05), * ( P ≤ 0.05), ** ( P ≤ 0.01), *** ( P ≤ 0.001), and **** ( P ≤ 0.0001). Scale bar in C = 50 µm. D Data showing that upregulation of PLCβ1 in PC12 cells causes the dissociation between TRBP and Egr-1 where TRBP was immunoprecipitated from PC12 cell lysates and Egr-1 levels were quantified by Western blotting. Bar graph shows mean values ± SD as well as individual data points. Unpaired t test was used to assess statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). E Immunofluorescence images probing changes in TRBP and Egr-1 in control HEK293 cells and cells induced to over-express PLCβ1. Scale bar in E = 50 µm.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: Transfection, Plasmid Preparation, Construct, Immunofluorescence, Immunoprecipitation, Western Blot, Control

Journal: Cell Death Discovery

Article Title: PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

doi: 10.1038/s41420-024-02009-z

Figure Lengend Snippet: At low PLCβ1 levels, Ago2 forms complexes that include TRBP [ , ] which binds Egr-1 in the cytosol [ , ] stabilizing the cytosolic localization of Egr-1. Increasing PLCβ1 helps solubilize Ago2 complexes releasing TRBP promoting nuclear localization of Egr-1, and potentially TRBP.

Article Snippet: For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102).

Techniques: