mouse sam68 gene orf cdna clone expression plasmid, c-flag tag (Sino Biological)

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Full length Clone DNA of Mouse KH domain containing, RNA binding, signal transduction associated 1 with C terminal Flag tag.

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his-tag/flag-tag sam68 (GenScript corporation)

GenScript corporation his-tag/flag-tag sam68

( A ) <t>Sam68</t> is a 443-amino acids protein. The QUA1 and KH domains are responsible for dimerization and RNA binding. The N-terminal region (residues 1-96), the QUA2 region (267-283) and the C-terminal region (residues 284-443) are predicted to be intrinsically disordered and contain regulatory motifs such as Pro-rich motifs (P0 to P5), RG-rich motifs (RG), a tyrosine-rich region (YY) and a nuclear localization sequence (NLS). The number and percentage of serine and threonine for each region/domain is indicated. ( B, C ) NMR ( 1 H- 15 N)-HSQC spectra of Sam68 N-term (residues 1-96) ( B ) and C-term (residues 267-368) at 4°C ( C ). The assignment of the backbone amide resonances is indicated.

His Tag/Flag Tag Sam68, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/flag+sam68/bio_rxiv__2022__03__23__485498-130-0-33?v=GenScript+corporation
Average 90 stars, based on 1 article reviews

his-tag/flag-tag sam68 - by Bioz Stars, 2026-08

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mouse sam68 gene orf cdna clone expression plasmid, n-flag tag (Sino Biological)

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Full length Clone DNA of Mouse KH domain containing, RNA binding, signal transduction associated 1 with N terminal Flag tag.

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( A ) Sam68 is a 443-amino acids protein. The QUA1 and KH domains are responsible for dimerization and RNA binding. The N-terminal region (residues 1-96), the QUA2 region (267-283) and the C-terminal region (residues 284-443) are predicted to be intrinsically disordered and contain regulatory motifs such as Pro-rich motifs (P0 to P5), RG-rich motifs (RG), a tyrosine-rich region (YY) and a nuclear localization sequence (NLS). The number and percentage of serine and threonine for each region/domain is indicated. ( B, C ) NMR ( 1 H- 15 N)-HSQC spectra of Sam68 N-term (residues 1-96) ( B ) and C-term (residues 267-368) at 4°C ( C ). The assignment of the backbone amide resonances is indicated.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: ( A ) Sam68 is a 443-amino acids protein. The QUA1 and KH domains are responsible for dimerization and RNA binding. The N-terminal region (residues 1-96), the QUA2 region (267-283) and the C-terminal region (residues 284-443) are predicted to be intrinsically disordered and contain regulatory motifs such as Pro-rich motifs (P0 to P5), RG-rich motifs (RG), a tyrosine-rich region (YY) and a nuclear localization sequence (NLS). The number and percentage of serine and threonine for each region/domain is indicated. ( B, C ) NMR ( 1 H- 15 N)-HSQC spectra of Sam68 N-term (residues 1-96) ( B ) and C-term (residues 267-368) at 4°C ( C ). The assignment of the backbone amide resonances is indicated.

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, Full-length GFP-Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (aa 1-96) into the pLEICS-01 vector and GST-Sam68 C-terminal region (267-368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: RNA Binding Assay, Sequencing

( A, B ) ( 1 H- 15 N)-HSQC spectra overlay of Sam68 N-term ( A ) and C-term ( B ) before (black) and after 16 hours incubation with commercial active Cdk-1/Cyclin B (red) at 4°C. T33 and T317 resonance peaks are indicated. ( C, D ) Chemical shift perturbation of backbone amides as a function of the N-term ( C ) and C-term ( D ) amino acid sequence upon Cdk1 phosphorylation. For clarity, the CSP of T33 and T317 are represented as red bars and are not to scale. ( E, F ) Normalized intensity of phosphorylated T33 ( E ) and T317 ( F ) backbone amide peak as a function of time after Cdk1/cyclin B addition. The data were fitted using a Hill function.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: ( A, B ) ( 1 H- 15 N)-HSQC spectra overlay of Sam68 N-term ( A ) and C-term ( B ) before (black) and after 16 hours incubation with commercial active Cdk-1/Cyclin B (red) at 4°C. T33 and T317 resonance peaks are indicated. ( C, D ) Chemical shift perturbation of backbone amides as a function of the N-term ( C ) and C-term ( D ) amino acid sequence upon Cdk1 phosphorylation. For clarity, the CSP of T33 and T317 are represented as red bars and are not to scale. ( E, F ) Normalized intensity of phosphorylated T33 ( E ) and T317 ( F ) backbone amide peak as a function of time after Cdk1/cyclin B addition. The data were fitted using a Hill function.

Techniques: Incubation, Sequencing, Phospho-proteomics

( A ) nucleotide sequence of the G8.5 RNA identified previously as a high-affinity binder of Sam68 . ( B, C ) ( 1 H- 15 N)-HSQC spectra of Sam68 N-term ( B ) and C-term ( C ) before (black) and after (red) addition of excess G8.5 RNA (protein:RNA molar ratio of 1:2) at 4°C. ( D, E ) Chemical shift perturbation of Sam68 N-term ( D ) and C-term ( E ) backbone resonances upon RNA interaction as a function of the amino acid sequence.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: ( A ) nucleotide sequence of the G8.5 RNA identified previously as a high-affinity binder of Sam68 . ( B, C ) ( 1 H- 15 N)-HSQC spectra of Sam68 N-term ( B ) and C-term ( C ) before (black) and after (red) addition of excess G8.5 RNA (protein:RNA molar ratio of 1:2) at 4°C. ( D, E ) Chemical shift perturbation of Sam68 N-term ( D ) and C-term ( E ) backbone resonances upon RNA interaction as a function of the amino acid sequence.

Techniques: Sequencing

( A, B ) ( 1 H- 15 N)-HSQC spectra of Cdk1-phosphorylated Sam68 N-term ( A ) and C-term ( B ) before (black) and after (red) addition of 2 molar equivalent of G8.5 RNA at 4°C. Resonances of T33 and T317 are indicated. ( C, D ) Chemical shift perturbation of Sam68 N-term ( C ) and C-term ( D ) in their unphosphorylated (black) or Cdk-1 phosphorylated (red) forms upon RNA binding. T33 and T317 CSPs are labeled with a green star.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: ( A, B ) ( 1 H- 15 N)-HSQC spectra of Cdk1-phosphorylated Sam68 N-term ( A ) and C-term ( B ) before (black) and after (red) addition of 2 molar equivalent of G8.5 RNA at 4°C. Resonances of T33 and T317 are indicated. ( C, D ) Chemical shift perturbation of Sam68 N-term ( C ) and C-term ( D ) in their unphosphorylated (black) or Cdk-1 phosphorylated (red) forms upon RNA binding. T33 and T317 CSPs are labeled with a green star.

Techniques: RNA Binding Assay, Labeling

( A, B ) Confocal fluorescence images of HEK-293T ( A ) and HCT-116 ( B ) cells transfected with either GFP-tagged Sam68 WT, or phospho-mimetic mutants. DAPI is shown in red and GFP in green. ( C ) Representative images of 3 different classes (patterns A-C) of Sam68 wild-type and mutants cellular localization in HCT-116 cells (Top) and percentage of cells displaying Sam68 or mutants localized in each class for 50-100 cells analysed per sample (Bottom).

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: ( A, B ) Confocal fluorescence images of HEK-293T ( A ) and HCT-116 ( B ) cells transfected with either GFP-tagged Sam68 WT, or phospho-mimetic mutants. DAPI is shown in red and GFP in green. ( C ) Representative images of 3 different classes (patterns A-C) of Sam68 wild-type and mutants cellular localization in HCT-116 cells (Top) and percentage of cells displaying Sam68 or mutants localized in each class for 50-100 cells analysed per sample (Bottom).

Techniques: Fluorescence, Transfection

Effect of Sam68 WT, T33E, T317E and T33E/T317E transfection on the alternative splicing of CD44 exon v5 ( A ), and Bcl-x ( B ) minigenes. Bottom: agarose gel electrophoresis showing splicing of the minigenes in response to co-transfected proteins. Top: quantification of biological replicates from three independent co-transfection experiments. Bar chart plotting and analysis were performed using GraphPad Prism. Error bars represent the standard deviation of three independent experiments. P values were calculated using an independent two-sample t-test (*: p<0.05, **: p<0.005 and ****: p<0.00005). Uncropped gels are shown in .

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: Effect of Sam68 WT, T33E, T317E and T33E/T317E transfection on the alternative splicing of CD44 exon v5 ( A ), and Bcl-x ( B ) minigenes. Bottom: agarose gel electrophoresis showing splicing of the minigenes in response to co-transfected proteins. Top: quantification of biological replicates from three independent co-transfection experiments. Bar chart plotting and analysis were performed using GraphPad Prism. Error bars represent the standard deviation of three independent experiments. P values were calculated using an independent two-sample t-test (*: p<0.05, **: p<0.005 and ****: p<0.00005). Uncropped gels are shown in .

Techniques: Transfection, Alternative Splicing, Agarose Gel Electrophoresis, Cotransfection, Standard Deviation

( A, B ) Effect of Sam68 WT and mutants transfection on cell cycle progression. ( A ) Flow cytometry images and ( B ) percentage of cells in sub G1 (black), G1/G0 (dark grey), S (white), and G2/M (light grey) phases 48 hours after transfection with Sam68 WT or mutants. ( C, D ) effect of Sam68 WT and mutants transfection on cell apoptosis. ( C ) Flow cytometry images and ( D ) percentage of apoptotic cells 48 hours after transfection with Sam68 WT or mutants. ( E ) Normalized cell proliferation increase between 48 and 72 hours after transfection with Sam68 WT or mutants. Bar chart represents the ratio between the number of GFP-positive cells at 72 hours over 48 hours. Error bars represent the standard deviation of three independent experiments. P values were calculated using an independent two-sample t-test (statistical significance shown as: *: p<0.05, **: p<0.005 and ***: p<0.0005).

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: ( A, B ) Effect of Sam68 WT and mutants transfection on cell cycle progression. ( A ) Flow cytometry images and ( B ) percentage of cells in sub G1 (black), G1/G0 (dark grey), S (white), and G2/M (light grey) phases 48 hours after transfection with Sam68 WT or mutants. ( C, D ) effect of Sam68 WT and mutants transfection on cell apoptosis. ( C ) Flow cytometry images and ( D ) percentage of apoptotic cells 48 hours after transfection with Sam68 WT or mutants. ( E ) Normalized cell proliferation increase between 48 and 72 hours after transfection with Sam68 WT or mutants. Bar chart represents the ratio between the number of GFP-positive cells at 72 hours over 48 hours. Error bars represent the standard deviation of three independent experiments. P values were calculated using an independent two-sample t-test (statistical significance shown as: *: p<0.05, **: p<0.005 and ***: p<0.0005).

Techniques: Transfection, Flow Cytometry, Standard Deviation

48 hours after transfection of HCT116 cells with Sam68 WT or mutants, the number of GFP(+) cells were counted every 2 hours for 24 hours with LiveCite 2 using Trainable Weka Segmentation 52 , ITCN plugin and macro in ImageJ 53 .

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: 48 hours after transfection of HCT116 cells with Sam68 WT or mutants, the number of GFP(+) cells were counted every 2 hours for 24 hours with LiveCite 2 using Trainable Weka Segmentation 52 , ITCN plugin and macro in ImageJ 53 .

Techniques: Transfection

Overlay spectra of N-term (left and C-term (right) regions of Sam68. A) resonances in the absence (black) or presence (green) of 2 molar equivalents of G8.5 RNA; B) resonances before (black) and after 8 hours incubation with Cdk1, MgCl 2 and ATP (red); C) resonances after Cdk1 phosphorylation in the absence (red) and presence (blue) of 2 molar equivalents of G8.5 RNA.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: Overlay spectra of N-term (left and C-term (right) regions of Sam68. A) resonances in the absence (black) or presence (green) of 2 molar equivalents of G8.5 RNA; B) resonances before (black) and after 8 hours incubation with Cdk1, MgCl 2 and ATP (red); C) resonances after Cdk1 phosphorylation in the absence (red) and presence (blue) of 2 molar equivalents of G8.5 RNA.

Techniques: Incubation, Phospho-proteomics

48 hours after transfection of HCT116 cells with Sam68 WT or mutants, the number of GFP(+) nad GFP(-) cells were counted. Cells were used for additional experiments only if more than 75% of cells were GFP positive.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: 48 hours after transfection of HCT116 cells with Sam68 WT or mutants, the number of GFP(+) nad GFP(-) cells were counted. Cells were used for additional experiments only if more than 75% of cells were GFP positive.

Techniques: Transfection

In interphase, Cdk-1 is inactive, so T33 and T317 of Sam68 are unphosphorylated. Sam68 would therefore bind RNA specifically through its QUA1-KH homodimerization domain with dissociation constant (Kd) in the low micromolar range and the N-terminal and C-terminal regions would anchor the protein to the RNA, increasing the affinity of full-length Sam68 to RNA (Kd in the low nanomolar range). During mitosis or in cancer cells, activation of Cdk-1/cyclin B induces the phosphorylation of T33 and T317, leading to the dissociation of the N-term and C-term anchoring region from the RNA and therefore, a weakening in RNA binding by full-length Sam68 making Sam68 less effective at competing with other splicing factors for RNA binding. As a consequence, Sam68 activity in splicing regulation is reduced, leading to a decrease in cell apoptosis and an increase in proliferation.

Journal: bioRxiv

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

doi: 10.1101/2022.03.23.485498

Figure Lengend Snippet: In interphase, Cdk-1 is inactive, so T33 and T317 of Sam68 are unphosphorylated. Sam68 would therefore bind RNA specifically through its QUA1-KH homodimerization domain with dissociation constant (Kd) in the low micromolar range and the N-terminal and C-terminal regions would anchor the protein to the RNA, increasing the affinity of full-length Sam68 to RNA (Kd in the low nanomolar range). During mitosis or in cancer cells, activation of Cdk-1/cyclin B induces the phosphorylation of T33 and T317, leading to the dissociation of the N-term and C-term anchoring region from the RNA and therefore, a weakening in RNA binding by full-length Sam68 making Sam68 less effective at competing with other splicing factors for RNA binding. As a consequence, Sam68 activity in splicing regulation is reduced, leading to a decrease in cell apoptosis and an increase in proliferation.

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

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