Journal: Archivum Immunologiae et Therapiae Experimentalis

Article Title: Detection and Significance of Cytotoxic Cell Subsets in Biopsies of HCV-Infected Human Livers

doi: 10.1007/s00005-013-0258-6

Figure Lengend Snippet: Primary antibodies used

Article Snippet: NKG2D/CD314 , Mouse , Monoclonal , 1D11 , Novus Biologicals , NB100-65956.

Techniques:

Journal: bioRxiv

Article Title: A frameshift mutation in the murine Prkra gene causes dystonia and exhibits abnormal cerebellar development and reduced eIF2α phosphorylation

doi: 10.1101/2024.06.04.597421

Figure Lengend Snippet: (A) dsRNA binding assay. dsRNA binding activity of WT PACT/RAX/RAX and lear-5J truncated protein was measured by a poly(I):poly(C)-agarose binding assay with in vitro translated 35 S-labeled proteins. T, total input; B, proteins bound to poly(I):poly(C)-agarose. Competition lanes 5 and 6: competition with 100-fold molar excess of single-stranded RNA (ss) or dsRNA (ds). The faint band below the parent PACT/RAX band represents products of in vitro translation from an internal methionine codon in lane 1. (B) Quantification of the dsRNA binding assay. Bands were quantified by phosphorimaging analysis, and % bound was calculated. Error bars: S.D. from three independent experiments. The p value (0.003) calculated using statistical analyses indicated significant difference between % dsRNA-binding of WT (blue bar) and lear-5J mutant (red bar) as indicated by the bracket. (C).Co-Immunoprecipitation of endogenous PKR and Flag-PACT/RAX or Flag-lear-5J overexpressed in HeLa cells. HeLa cells were transfected with Flag wt PACT/RAX or Flag-lear-5J in pCDNA3.1-expression constructs at 40% confluency and harvested 24-hour post transfection. Whole cell extracts were immunoprecipitated at 4°C overnight using 100 ng of anti-PKR antibody per IP. Samples were then analyzed via SDS-PAGE gel electrophoresis and western blot analysis probing for Flag tagged wt PACT/RAX or lear-5J (co-IP panel) using monoclonal anti-Flag-M2 (Sigma) antibody. To ascertain that an equal amount of protein was immunoprecipitated, blots were re-probed using an anti-PKR antibody (IP panel). Input blots without immunoprecipitation demonstrate equal amounts of each protein were present prior to IP.

Article Snippet: Whole cell extract was then immunoprecipitated overnight at 4°C on a rotating wheel using anti-PKR antibody (71/10, R&D Systems) conjugated to protein A sepharose beads (GE Healthcare) in IP buffer.

Techniques: Binding Assay, Activity Assay, In Vitro, Labeling, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Construct, SDS Page, Nucleic Acid Electrophoresis, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: A frameshift mutation in the murine Prkra gene causes dystonia and exhibits abnormal cerebellar development and reduced eIF2α phosphorylation

doi: 10.1101/2024.06.04.597421

Figure Lengend Snippet: (A) Effect of lear-5J protein on PKR kinase activity. PKR kinase activity assay was performed using PKR immunoprecipitated from HeLa cells, recombinant lear-5J and wt PACT/RAX proteins, and 1 μCi of [γ- 32 P] ATP per reaction. Either pure recombinant lear-5J (left) or wt PACT/RAX (right) protein was added as activator in amounts indicated above each lane. Phosphorylated PKR was visualized after SDS-PAGE and phosphorimager analysis. (B) The truncated lear-5J protein inhibits PKR activation. PKR immunoprecipitated from HeLa cell extracts was activated with of polyI:polyC (lanes 2-5) or 4 ng of recombinant pure wt PACT/RAX protein (lanes 6-9). Increasing amounts of recombinant pure lear-5J protein (4 ng-400 ng) were added (lanes 2-9) as indicated to assess the effect on PKR activity. Lane 1: PKR activity without any added activator, lane 2: PKR activity in the presence of polyI:polyC, lanes 3-5: PKR activity in the presence of polyI:polyC and 4ng, 40 ng, or 400 ng of lear-5J protein, lane 6: PKR activity in the presence of 4ng of wt PACT/RAX protein, lanes 7-9: PKR activity in the presence of 4 ng of wt PACT/RAX and 4 ng, 40 ng or 400 ng of lear-5J protein. Phosphorylated proteins were analyzed by SDS-PAGE and phosphorimager analysis.

Article Snippet: Whole cell extract was then immunoprecipitated overnight at 4°C on a rotating wheel using anti-PKR antibody (71/10, R&D Systems) conjugated to protein A sepharose beads (GE Healthcare) in IP buffer.

Techniques: Activity Assay, Kinase Assay, Immunoprecipitation, Recombinant, SDS Page, Activation Assay

Journal: Cell Death & Disease

Article Title: An interplay between UCP2 and ROS protects cells from high-salt-induced injury through autophagy stimulation

doi: 10.1038/s41419-021-04188-4

Figure Lengend Snippet: FACS analysis of microvascular endothelial cells silenced for UCP2 (siUCP2) and treated for 72 h with 20 mM NaCl either in the presence or in the absence of TAT-Beclin D11 (10 μM) ( N = 6). Representative scatter plots ( A ) and quantification of live, apoptotic, and necrotic cells ( B ) are reported. CTR indicates not transfected and not treated cells. ** p < 0.01, *** p < 0.001 obtained using the one-way ANOVA followed by Bonferroni post hoc analysis. Data are reported as mean ± SEM.

Article Snippet: FACS analysis was performed also in cells treated with the enhancer of autophagy TAT-Beclin D11 (NBP2-49888, Novus Biological, Centennial, Colorado, US).

Techniques: Transfection

Journal: Cell Death & Disease

Article Title: An interplay between UCP2 and ROS protects cells from high-salt-induced injury through autophagy stimulation

doi: 10.1038/s41419-021-04188-4

Figure Lengend Snippet: FACS analysis of renal proximal tubular epithelial cells silenced for UCP2 (siUCP2) and treated for 72 h with 20 mM NaCl either in the presence or in the absence of TAT-Beclin D11 (10 μM) ( N = 6). Representative scatter plots ( A ) and quantification of live, apoptotic, and necrotic cells ( B ) are reported. CTR indicates not transfected and not treated cells. ** p < 0.01, *** p < 0.001 obtained using the one-way ANOVA followed by Bonferroni post hoc analysis. Data are reported as mean ± SEM.

Article Snippet: FACS analysis was performed also in cells treated with the enhancer of autophagy TAT-Beclin D11 (NBP2-49888, Novus Biological, Centennial, Colorado, US).

Techniques: Transfection