Journal:

Article Title: Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21

doi: 10.1073/pnas.0403130101

Figure Lengend Snippet: hRAD9 and p53 regulate p21 RNA and encoded protein levels. (A) Northern analysis of p21 expression. Exponentially growing H1299 cells were transfected with equal amounts (1 μg) of indicated plasmids and harvested for isolation of total RNA 48 h later. RNA (20 μg) was fractionated on a 1.2% formaldehyde agarose gel and transferred onto a nylon membrane for Northern blotting by using a [α-32P]dCTP-labeled human p21 probe following standard procedures. The blot was rehybridized with a human GAPDH cDNA probe as a loading control. Cells were transfected with: lane 1, pcDNA3; lane 2, pZeoSV2(+); lane 3, pZeoSV2(+)-RAD9; lane 4, pC53-SN3; lane 5, pZeoSV2(+)-RAD9 and pC53-SN3. (B) Western analysis of p21 protein. H1299 cells were transiently transfected, and cell-free protein extracts were prepared 48 h later. Equal amounts (40 μg) of each preparation were loaded onto a SDS-polyacrylamide gel for standard Western analysis using antibodies against p21, hRAD9, p53, or actin. H1299 cells were transfected with the following vectors: lane 1, pcDNA3; lane 2, pC53-SN3; lane 3, pZeoSV2(+)-RAD9; lane 4, pC53-SN3 and pZeoSV2(+)-RAD9; lane 5, pZeoSV2(+); lane 6, C32 whole cell lysate (Santa Cruz Biotechnology) control.

Article Snippet: Oligos with various lengths of p53 consensus sequence were used to confirm the minimum sequence necessary and identify critical sequences for hRAD9 binding.

Techniques: Northern Blot, Expressing, Transfection, Isolation, Agarose Gel Electrophoresis, Membrane, Labeling, Control, Western Blot

Journal:

Article Title: Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21

doi: 10.1073/pnas.0403130101

Figure Lengend Snippet: hRAD9 and p53 regulate p21 promoter activity in a transient expression assay. H1299 cells were transiently transfected with pcDNA3, pZeoSV2(+), pZeoSV2(+)-RAD9, or pC53-SN3, together with pGL2-p21-Luci, a luciferase reporter with the human p21 promoter upstream in pGL2. Extracts were assayed for luciferase activity on a Berthold Autolumat LB953 Rack Luminometer. All luciferase measurements are expressed as means ± SD of triplicate transfections and cultures. Transfection groups include the following (from left to right): insertless pcDNA3 vector and pGL2-basic; insertless pZeoSV2(+) vector and pGL2-basic; pZeoSV2(+) and pGL2-p21-Luci; pZeoSV2(+)-RAD9 and pGL2-p21-Luci; pC53-SN3 and pGL2-p21-Luci; (6) pZeoSV2(+)-RAD9 and pC53-SN3 and pGL2-p21-Luci.

Article Snippet: Oligos with various lengths of p53 consensus sequence were used to confirm the minimum sequence necessary and identify critical sequences for hRAD9 binding.

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation

Journal:

Article Title: Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21

doi: 10.1073/pnas.0403130101

Figure Lengend Snippet: Identification of a common binding site for hRAD9 and p53 in the p21 promoter. (A) hRAD9 binds to oligos containing a p53 consensus site in the p21 promoter. 32P-labeled oligos were incubated with nuclear protein extract from growing U937 cells. Resulting protein–DNA complexes were resolved by 4% acrylamide gel electrophoresis and visualized by exposing x-ray film. Three gel-shift bands were formed in lane 1 as indicated. Cold competitor oligos were present in 100-fold excess to demonstrate specificity of the protein–DNA complexes (lanes 2 and 4). Anti-RAD9 antibodies were added to identify proteins in the complexes (lanes 3 and 4). Oligos with various lengths of p53 consensus sequence were used to confirm the minimum sequence necessary and identify critical sequences for hRAD9 binding. Lines indicate protein-antibody–DNA complex (Upper) and protein/DNA complex (Lower). (B) p53 binds to oligos containing the hRAD9 binding element. Human p53 protein (20) was incubated with 32P-labeled oligos and resolved by gel electrophoresis. Unlabeled competitor oligo was added in 100-fold excess for lane 2. Anti-p53 antibodies were used for the supershift in lane 3. Wild-type and modified probes were used as above (A). (C) Oligos used for EMSA. Wild-type (30-bp) and mutated (30-bp) oligo differ by 1 bp (lowercase letter). Deleted oligos were designed to omit base pairs at the ends of the starting 30-mer.

Article Snippet: Oligos with various lengths of p53 consensus sequence were used to confirm the minimum sequence necessary and identify critical sequences for hRAD9 binding.

Techniques: Binding Assay, Labeling, Incubation, Acrylamide Gel Assay, Electrophoresis, Gel Shift, Sequencing, Nucleic Acid Electrophoresis, Modification

Journal: Journal of Virology

Article Title: Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories

doi: 10.1128/JVI.01110-19

Figure Lengend Snippet: Viral RNA detection in rRV-NSP5/S67A-infected cells. (A) Representative confocal immunofluorescence micrographs of MA104 (upper panel), MA-NSP2-mCherry (middle panel), and MA-NSP5 (lower panel) cells infected with rRV-wt or rRV-NSP5/S67A strains and stained with anti-NSP5 (red in MA104 and MA-NSP5 cells) and 5-EU (green). (B) Single-molecule RNA fluorescence in situ hybridization (smFISH) of MA-NSP5-EGFP cells infected with rRV-wt, rRV-NSP5/S67A, or rRV-NSP5/ΔT strains. Viroplasms detected with NSP5-EGFP (green) and viral RNA (the probe specific for gs6 was Cy3 conjugated; red). Colocalizing viroplasms and RNAs are indicated by white arrows. Scale bar, 15 μm. (C) Quantitative analysis of NSP5-EGFP-positive structures (viroplasms) for RNA (gs6) in MA-NSP5-EGFP cells infected with rRV-wt or rRV-NSP5/S67A. ***, P < 0.001.

Article Snippet: Rehydrated samples were hybridized with a 62.5 nM concentration of an equimolar mixture of Cy3-labeled DNA probes designed to target the coding region of gene segment 6 of simian rotavirus A/SA11 (GenBank accession no. AY187029.1 ) using Stellaris Probe Designer v2 software (LCG Biosearch Technologies), in a total volume of 200 μl of the hybridization buffer (Stellaris RNA fluorescence in situ hybridization [FISH] hybridization buffer, SMF-HB1-10 [Biosearch Technologies], supplemented with 10% [vol/vol] deionized formamide).

Techniques: RNA Detection, Infection, Immunofluorescence, Staining, Fluorescence, In Situ Hybridization