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sam68 expression plasmids pgex 2 t sam68  (Addgene inc)


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    Addgene inc sam68 expression plasmids pgex 2 t sam68
    Fig. 1 <t>Sam68</t> redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left; and IBRS2, right) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 (red). Nuclei were stained with DAPI (blue)
    Sam68 Expression Plasmids Pgex 2 T Sam68, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sam68+expression+plasmids+pgex+2+t+sam68/pcDNA3+HA+Sam68+WT+(Plasmid+%2317690)/pm26695943-266-0-21
    Average 93 stars, based on 9 article reviews
    sam68 expression plasmids pgex 2 t sam68 - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections."

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

    Journal: Virology journal

    doi: 10.1186/s12985-015-0452-8

    Fig. 1 Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left; and IBRS2, right) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 (red). Nuclei were stained with DAPI (blue)
    Figure Legend Snippet: Fig. 1 Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left; and IBRS2, right) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 (red). Nuclei were stained with DAPI (blue)

    Techniques Used: Infection, Staining

    Fig. 2 FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 (a) or mouse monoclonal anti-G3BP (b) followed by donkey-anti-goat-AF568 (red, a) or goat-anti- mouse-AF568 (red; b). Nuclei were stained with DAPI (blue)
    Figure Legend Snippet: Fig. 2 FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 (a) or mouse monoclonal anti-G3BP (b) followed by donkey-anti-goat-AF568 (red, a) or goat-anti- mouse-AF568 (red; b). Nuclei were stained with DAPI (blue)

    Techniques Used: Infection, Staining

    Fig. 3 Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section (b). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section (b) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank
    Figure Legend Snippet: Fig. 3 Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section (b). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section (b) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank

    Techniques Used: Sequencing, Binding Assay, Western Blot, Labeling, Mutagenesis, Construct, Control

    Fig. 4 Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted (left lane) and depleted (right lane) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A24-Cru. FMDV A24-Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3Dpol. c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A24-Cru RNA. FMDV A24-Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting
    Figure Legend Snippet: Fig. 4 Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted (left lane) and depleted (right lane) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A24-Cru. FMDV A24-Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3Dpol. c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A24-Cru RNA. FMDV A24-Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting

    Techniques Used: Western Blot, SDS Page

    Fig. 6 Sam68 interacts with FMDV 3Cpro and 3Dpol. a Co-immunoprecipitation of FMDV 3Dpol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3Dpol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3Dpol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3Dpol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3Dpol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3Dpol, starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3Dpol and Sam68. (i) Electrostatic surface representation of FMDV 3Dpol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3Dpol. (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3Dpol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3Dpol. [98] FMDV 3Dpol green docked on Sam68 blue in cartoon representation. The 3Dpol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3Dpol (d) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3Cpro, and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane
    Figure Legend Snippet: Fig. 6 Sam68 interacts with FMDV 3Cpro and 3Dpol. a Co-immunoprecipitation of FMDV 3Dpol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3Dpol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3Dpol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3Dpol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3Dpol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3Dpol, starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3Dpol and Sam68. (i) Electrostatic surface representation of FMDV 3Dpol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3Dpol. (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3Dpol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3Dpol. [98] FMDV 3Dpol green docked on Sam68 blue in cartoon representation. The 3Dpol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3Dpol (d) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3Cpro, and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane

    Techniques Used: Immunoprecipitation, Infection, Western Blot, Control, Sequencing, Negative Control, Binding Assay

    Related Articles

    Expressing:

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.
    Article Snippet: Lipofectamine 2000 DNA transfection reagent and streptavidin-coupled magnetic beads were purchased from Invitrogen (Carlsbad, CA). .. Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA. .. LightShift® Chemiluminescent RNA EMSA Kit and the NE-PER cellular fractionation kit were purchased from Thermo Fisher Scientific Inc. (Rockford, IL).

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections
    Article Snippet: Lipofectamine 2000 DNA transfection reagent and streptavidin-coupled magnetic beads were purchased from Invitrogen (Carlsbad, CA). .. Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA. .. LightShift® Chemiluminescent RNA EMSA Kit and the NE-PER cellular fractionation kit were purchased from Thermo Fisher Scientific Inc. (Rockford, IL).



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    sam68 expression plasmids pgex 2 t sam68  (Addgene inc)
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    Addgene inc sam68 expression plasmids pgex 2 t sam68
    Fig. 1 <t>Sam68</t> redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left; and IBRS2, right) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 (red). Nuclei were stained with DAPI (blue)
    Sam68 Expression Plasmids Pgex 2 T Sam68, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sam68+expression+plasmids+pgex+2+t+sam68/pcDNA3+HA+Sam68+WT+(Plasmid+%2317690)/pm26695943-266-0-21
    Average 93 stars, based on 1 article reviews
    sam68 expression plasmids pgex 2 t sam68 - by Bioz Stars, 2026-09
    93/100 stars
    		  Buy from Supplier

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    Fig. 1 Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left; and IBRS2, right) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 (red). Nuclei were stained with DAPI (blue)

    Journal: Virology journal

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

    doi: 10.1186/s12985-015-0452-8

    Figure Lengend Snippet: Fig. 1 Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left; and IBRS2, right) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 (red). Nuclei were stained with DAPI (blue)

    Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

    Techniques: Infection, Staining

    Fig. 2 FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 (a) or mouse monoclonal anti-G3BP (b) followed by donkey-anti-goat-AF568 (red, a) or goat-anti- mouse-AF568 (red; b). Nuclei were stained with DAPI (blue)

    Journal: Virology journal

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

    doi: 10.1186/s12985-015-0452-8

    Figure Lengend Snippet: Fig. 2 FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 (a) or mouse monoclonal anti-G3BP (b) followed by donkey-anti-goat-AF568 (red, a) or goat-anti- mouse-AF568 (red; b). Nuclei were stained with DAPI (blue)

    Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

    Techniques: Infection, Staining

    Fig. 3 Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section (b). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section (b) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank

    Journal: Virology journal

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

    doi: 10.1186/s12985-015-0452-8

    Figure Lengend Snippet: Fig. 3 Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section (b). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section (b) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank

    Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

    Techniques: Sequencing, Binding Assay, Western Blot, Labeling, Mutagenesis, Construct, Control

    Fig. 4 Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted (left lane) and depleted (right lane) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A24-Cru. FMDV A24-Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3Dpol. c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A24-Cru RNA. FMDV A24-Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting

    Journal: Virology journal

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

    doi: 10.1186/s12985-015-0452-8

    Figure Lengend Snippet: Fig. 4 Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted (left lane) and depleted (right lane) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A24-Cru. FMDV A24-Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3Dpol. c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A24-Cru RNA. FMDV A24-Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting

    Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

    Techniques: Western Blot, SDS Page

    Fig. 6 Sam68 interacts with FMDV 3Cpro and 3Dpol. a Co-immunoprecipitation of FMDV 3Dpol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3Dpol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3Dpol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3Dpol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3Dpol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3Dpol, starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3Dpol and Sam68. (i) Electrostatic surface representation of FMDV 3Dpol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3Dpol. (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3Dpol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3Dpol. [98] FMDV 3Dpol green docked on Sam68 blue in cartoon representation. The 3Dpol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3Dpol (d) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3Cpro, and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane

    Journal: Virology journal

    Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

    doi: 10.1186/s12985-015-0452-8

    Figure Lengend Snippet: Fig. 6 Sam68 interacts with FMDV 3Cpro and 3Dpol. a Co-immunoprecipitation of FMDV 3Dpol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3Dpol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3Dpol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3Dpol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3Dpol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3Dpol, starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3Dpol and Sam68. (i) Electrostatic surface representation of FMDV 3Dpol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3Dpol. (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3Dpol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3Dpol. [98] FMDV 3Dpol green docked on Sam68 blue in cartoon representation. The 3Dpol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3Dpol (d) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3Cpro, and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane

    Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

    Techniques: Immunoprecipitation, Infection, Western Blot, Control, Sequencing, Negative Control, Binding Assay