Journal:

Article Title: RelA-Associated Inhibitor Blocks Transcription of Human Immunodeficiency Virus Type 1 by Inhibiting NF-?B and Sp1 Actions

doi: 10.1128/JVI.76.16.8019-8030.2002

Figure Lengend Snippet: Binding of Sp1 and RAI in vivo. (A) Schematic diagram of RAI plasmids: wild-type RAI (containing ankyrin repeats and the SH3 domain), RAI ΔN (containing 132 to 351 amino acids), and RAI ΔC (containing 1 to 146 amino acids). (B and C) The lysates prepared from 293 cells transfected with pFLAG (empty vector), pFLAG-RAI (wild type), pFLAG-RAIΔC, or pFLAG-RAIΔN were used for coimmunoprecipitation. Whole-cell lysates were prepared from 293 cells 24 h after transfection and were precipitated with anti-FLAG affinity gel. Immune complexes were collected and subjected to SDS-PAGE followed by Western blotting with anti-Sp1 antibody. The position of the Sp1 proteins and Ig heavy chain (IgH) proteins are indicated. Sp1 was detected in the lysate overexpressing FLAG-RAI wild type and FLAG-RAI ΔN. As input, 1/20 of the proteins used in each reaction was loaded.

Article Snippet: Briefly, an equal amount of the recombinant Sp1 or MBP-p65 was incubated with 5 μg of the GST-RAI or GST (as a negative control) bound to glutathione-Sepharose beads and in 250 μl of buffer A ( 39 ) at 4°C for 12 h. The beads were washed five times, and the bound proteins were eluted with an equal volume of 2× sodium dodecyl sulfate (SDS) loading buffer, boiled for 3 min, and resolved by SDS-8% polyacrylamide gel electrophoresis (SDS-8% PAGE) followed by Western blotting with goat anti-Sp1 (PEP 2) antibody or rabbit anti-p65 antibody (Santa Cruz) at a dilution of 1/1,000.

Techniques: Binding Assay, In Vivo, Transfection, Plasmid Preparation, SDS Page, Western Blot

Journal: Molecular cell

Article Title: Multisite phosphorylation of S6K1 directs a kinase phospho-code that determines substrate selection

doi: 10.1016/j.molcel.2018.11.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunoblot analysis Ccell lysates or immunoprecipitates were denatured in Laemmli sample buffer (Bio-Rad) and resolved on Tris-glycine SDS-PAGE (10, 12, or 15% polyacrylamide) prepared using 37.5:1 acrylamide:bis-acrylamide stock solution (National Diagnostics), or on precast Express Plus Tris-MOPS-SDS PAGE (8, 10, 12, 4-12%, 4-20%; GenScript).

Techniques: Virus, Recombinant, Purification, Mutagenesis, Cell Culture, Extraction, Staining, Protease Inhibitor, Western Blot, Silver Staining, Transfection, Affinity Chromatography, Plasmid Preparation, Control, Software