polyclonal rabbit anti rpl7a  (Danaher Inc)

Journal: Journal of Virology

Article Title: Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

doi: 10.1128/JVI.03443-13

Figure Lengend Snippet: Mass spectrometry-based identification and quantification of proteins interacting with PARP12

Article Snippet: Pellets were resuspended in protein loading buffer and analyzed by Western blotting using anti-Flag M2 antibodies (Sigma-Aldrich), polyclonal rabbit anti-RPL7a antibodies (Bethyl), and infrared dye-labeled secondary antibodies and scanned on a Li-Cor imager.

Techniques:

Journal: Journal of Virology

Article Title: Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

doi: 10.1128/JVI.03443-13

Figure Lengend Snippet: Upon expression in BHK-21 cells, the long isoform of PARP12 (PARP12L) forms two types of complexes. (A) BHK-21 cells were infected with packaged replicon VEErep/PARP12L at an MOI of 20 infectious units per cell. At 4 h postinfection, cells were harvested and lysed with NP-40. After pelleting the nuclei, the lysates were either directly analyzed by ultracentrifugation in the sucrose gradients and fractionated as described in Materials and Methods or additionally incubated prior to ultracentrifugation in the presence of 10 mM EDTA to disassemble ribosomes. Ribosomes and their subunits and other protein complexes were pelleted by another round of ultracentrifugation and further analyzed by quantitative Western blotting using Flag-specific MAbs and antibodies specific to the ribosomal RPL7a protein, which is located in the 60S ribosomal subunit. Images were acquired and processed using a Li-Cor imager. The lower panel presents the results of quantitative analysis of Flag-PARP12L distribution in the sucrose gradient. The intensity of the signal in each fraction was normalized to the highest concentration of Flag-PARP12L. (B) Cells were infected and lysates were analyzed as described above except that before ultracentrifugation, they were treated with 4 μg/ml of RNase A to destroy polysomes. The lower panel presents the results of quantitative analysis of PARP12L and 60S ribosomal subunit distribution in the sucrose gradients. The intensity of the signal in each fraction was normalized to the signal detected in the fractions containing highest concentration of Flag-PARP12L or RPL7a. These experiments were repeated twice with essentially the same results.

Article Snippet: Pellets were resuspended in protein loading buffer and analyzed by Western blotting using anti-Flag M2 antibodies (Sigma-Aldrich), polyclonal rabbit anti-RPL7a antibodies (Bethyl), and infrared dye-labeled secondary antibodies and scanned on a Li-Cor imager.

Techniques: Expressing, Infection, Incubation, Western Blot, Concentration Assay

Journal: Journal of Virology

Article Title: Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

doi: 10.1128/JVI.03443-13

Figure Lengend Snippet: Expression of PARP12L, but not replication of VEErep itself, leads to destruction of polysomes. (A) BHK-21 cells were infected with packaged replicon VEErep/PARP12L at an MOI of 20 infectious units per cell. At 12 h postinfection, cells were harvested and lysed with NP-40, and the lysate was analyzed without additional treatment by ultracentrifugation in a sucrose gradient. Ribosomes, their subunits, and other protein complexes were pelleted from the fractions by additional ultracentrifugation and further analyzed by quantitative Western blotting using Flag-specific MAbs and antibodies specific to ribosomal RPL7a protein. Images were acquired and processed on a Li-Cor imager. The lower panel presents the results of quantitative analysis of Flag-PARP12L and 60S ribosomal subunit distribution in the sucrose gradient. The data were normalized to the signals detected in the fractions containing the highest concentrations of Flag-PARP12L and 60S subunit. (B) BHK-21 cells were infected with packaged VEErep/PARPrev replicon at an MOI of 20 infectious units per cell. At 4 and 12 h postinfection, cells were harvested and lysed with NP-40, and the lysates were analyzed without additional treatment by ultracentrifugation as described above for panel A.

Article Snippet: Pellets were resuspended in protein loading buffer and analyzed by Western blotting using anti-Flag M2 antibodies (Sigma-Aldrich), polyclonal rabbit anti-RPL7a antibodies (Bethyl), and infrared dye-labeled secondary antibodies and scanned on a Li-Cor imager.

Techniques: Expressing, Infection, Western Blot

Journal: Journal of Virology

Article Title: Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

doi: 10.1128/JVI.03443-13

Figure Lengend Snippet: The amino-terminal (PARP12-N) and the carboxy-terminal (PARP-C) domains of PARP12L form different complexes. BHK-21 cells were infected with packaged replicon VEErep/PARP12-C (A) or VEErep/PARP12-N (B) at an MOI of 20 infectious units per cell. At 4 h postinfection, cells were harvested and lysed with NP-40. After pelleting the nuclei, the lysates were either directly analyzed by ultracentrifugation in the sucrose gradients and fractionated as described in Materials and Methods or additionally incubated in the presence of 10 mM EDTA to disassemble ribosomes or with RNase A at concentration of 4 μg/ml for 15 min on ice to degrade polysomes. Ribosomes and their subunits and other protein complexes were pelleted by another round of ultracentrifugation as described in Materials and Methods and further analyzed by quantitative Western blotting using Flag-specific MAbs and antibodies specific to ribosomal RPL7a protein. Images were acquired and processed using a Li-Cor imager. The lower panels present the results of quantitative analysis of Flag-PARP12-C and Flag-PARP12-N distribution in the sucrose gradient. The intensity of the signal in each fraction was normalized to the highest concentration of Flag-PARP12L.

Article Snippet: Pellets were resuspended in protein loading buffer and analyzed by Western blotting using anti-Flag M2 antibodies (Sigma-Aldrich), polyclonal rabbit anti-RPL7a antibodies (Bethyl), and infrared dye-labeled secondary antibodies and scanned on a Li-Cor imager.

Techniques: Infection, Incubation, Concentration Assay, Western Blot

Journal: Journal of Virology

Article Title: Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

doi: 10.1128/JVI.03443-13

Figure Lengend Snippet: Mutation in PARP12L catalytic site does not abrogate formation of protein complexes. BHK-21 cells were infected with packaged VEErep/PARP12L-mCat replicon at an MOI of 20 infectious units per cell. At 4 h postinfection, cells were harvested and lysed with NP-40. After pelleting the nuclei, the lysates were either directly analyzed by ultracentrifugation in sucrose gradients and fractionated as described in Materials and Methods or additionally incubated in the presence of 10 mM EDTA to disassemble ribosomes. Ribosomes and their subunits and other protein complexes were pelleted by another round of ultracentrifugation as described in Materials and Methods and further analyzed by quantitative Western blotting using Flag-specific MAbs and antibodies specific to ribosomal RPL7a protein. Images were acquired and processed using a Li-COR imager. The lower panel presents the results of quantitative analysis of Flag-PARP12L-mCat distribution in the sucrose gradient. The intensity of the signal in each fraction was normalized to the highest concentration of Flag-PARP12L-mCat.

Article Snippet: Pellets were resuspended in protein loading buffer and analyzed by Western blotting using anti-Flag M2 antibodies (Sigma-Aldrich), polyclonal rabbit anti-RPL7a antibodies (Bethyl), and infrared dye-labeled secondary antibodies and scanned on a Li-Cor imager.

Techniques: Mutagenesis, Infection, Incubation, Western Blot, Concentration Assay