Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein

doi: 10.1073/pnas.1509745112

Figure Lengend Snippet: PB2 and PA are poly(ADP-ribosylated), but not by ZAPL, and this poly(ADP-ribosylation) is involved in ZAPL-mediated degradation. (A) The 293T cells were transfected with the indicated plasmids and then treated with MG132 where indicated. Cell extracts were immunoprecipitated with Flag Ab, followed by immunoblots probed with pADPr Ab. (B) V5-Z1 was overexpressed in 293T cells followed by retrovirus expression of Flag-PB2 and then MG132 treatment. The level of poly(ADP-ribosylation) of PB2 was determined as described in A. (C) The 293T cells were transfected with a PB2 plasmid and a Flag-Z2 or empty plasmid followed by MG132 treatment. Extracts were immunoprecipitated with Flag Ab, followed by immunoblots probed with pADPr or PB2 Ab. (D) PARG was knocked down in 293T cells by a siRNA to determine the effect on the level of retrovirus-expressed PB2 as described in SI Materials and Methods. See also Fig. S5.

Article Snippet: The eluate was heated at 70 °C in a buffer containing 60 mM Tris⋅HCl 6.8, 2% (vol/vol) SDS, 10% (vol/vol) glycerol, and 1.25% (vol/vol) β-mercaptoethanol and was analyzed by immunoblots probed with an Ab against pADPr (LP96-10, BD-Pharmingen) ( 19 , 31 ) or the Ab against the indicated protein.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein

doi: 10.1073/pnas.1509745112

Figure Lengend Snippet: Verification that PB2 and PA are poly(ADP-ribosylated). The 293T cells were transfected with the indicated plasmids and then treated with MG132 where indicated. Cell extracts were subjected to denaturing conditions to dissociate PB2 and PA from other proteins. The extracts were then immunoprecipitated with Flag Ab, followed by immunoblots probed with pADPr Ab. This figure is related to Fig. 5.

Article Snippet: The eluate was heated at 70 °C in a buffer containing 60 mM Tris⋅HCl 6.8, 2% (vol/vol) SDS, 10% (vol/vol) glycerol, and 1.25% (vol/vol) β-mercaptoethanol and was analyzed by immunoblots probed with an Ab against pADPr (LP96-10, BD-Pharmingen) ( 19 , 31 ) or the Ab against the indicated protein.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: Assessing kinetics and recruitment of DNA repair factors using high content screens

doi: 10.1016/j.celrep.2021.110176

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-PAR , Enzo Lab Sciences , Cat# ALX-210-890A-0100.

Techniques: Recombinant, Transfection, Electron Microscopy, Bicinchoninic Acid Protein Assay, Control, Negative Control, esiRNA, Plasmid Preparation, Software, High Content Screening, Microscopy, Cell Culture, Imaging