cdk9 cyclin t1  (Carna Inc)

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: HIV-1 Transcription Inhibition Using Small RNA-Binding Molecules.

doi: 10.3390/ph17010033

Figure Lengend Snippet: Figure 4. Disruption of Tat–TAR RNA interaction and change in RNA–protein interaction dynamics by 110FA and 102FA. (A) Structures of Wild-type (WT) TAR RNA, Delta TAR RNA (with Tat- binding bulge deleted), and Gamma TAR RNA (Cyclin T1 binding loop deleted) linked to the Biotin moiety. (B) Biotinylated RNA pulldown assay followed by immunoblot using protein lysate prepared from 293T cells expressing Flag-Tat and Biotin-WT TAR RNA or mutants coupled to beads. After incubation, the mixture was treated with 10 µM of the TAR RNA-binding molecules 110FA and 102FA. Coupled proteins were eluted from beads with Laemmli buffer, run on SDS-PAGE and detected with Anti-Flag, HEXIM-1, Cyclin T1, and Cdk9 antibodies.

Article Snippet: Primary antibody in PBS-T was added to the membranes prior to overnight incubation at 4 ◦C; primary antibodies include: α-Flag M2 (Cat: F1804; Sigma Aldrich, St. Louis, MO, USA, 1:1000), α-HEXIM1 (Cat: 12604; Cell Signaling Technology, Danvers, MA, USA, 1:1000), α-Cyclin T1 (Cat: 81464, Cell signaling Technology, 1:1000), α-Cdk9 (Cat: 2316, Cell signaling Technology, 1:1000) and β-actin (Cat: ab-49900; Abcam, Waltham, MA, USA, 1:5000), α-p-Cdk9 (T-186) (Cat: ab79178, Abcam, 1:1000), gp120, α-p24 and α-Nef were obtained from NIH AIDS Reagent Program, Manassas, VA, USA.

Techniques: Disruption, Binding Assay, Western Blot, Expressing, Incubation, RNA Binding Assay, SDS Page

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: HIV-1 Transcription Inhibition Using Small RNA-Binding Molecules.

doi: 10.3390/ph17010033

Figure Lengend Snippet: Figure 6. Effect of TAR-binding molecules on Tat-associated Cyclin T1 and Cdk9 protein levels. (A) HEK293T cells were transfected with the Flag-Tat plasmid (24 h) followed by TAR-binding molecules (110FA, 102FA) treatment at 1 µM concentration for 24 h, and cell lysate was collected. Cell lysates were incubated with protein A/G beads (pre-blocked with BSA), followed by immunopre- cipitation with Anti-Flag antibody and IgG as a control overnight at 4 ◦C. Beads were then washed, Tat-associated proteins were eluted from beads using Laemmli buffer, and SDS-PAGE was run. Im- munoblot was probed against the antibodies Cyclin T1 and Cdk9. Densitometry counts shown are results from three independent experiments with * p = 0.04 and ** p = 0.001 in comparison to the DMSO controls as shown in (B,C) for proteins Cyclin T1 and Cdk9, respectively.

Article Snippet: Primary antibody in PBS-T was added to the membranes prior to overnight incubation at 4 ◦C; primary antibodies include: α-Flag M2 (Cat: F1804; Sigma Aldrich, St. Louis, MO, USA, 1:1000), α-HEXIM1 (Cat: 12604; Cell Signaling Technology, Danvers, MA, USA, 1:1000), α-Cyclin T1 (Cat: 81464, Cell signaling Technology, 1:1000), α-Cdk9 (Cat: 2316, Cell signaling Technology, 1:1000) and β-actin (Cat: ab-49900; Abcam, Waltham, MA, USA, 1:5000), α-p-Cdk9 (T-186) (Cat: ab79178, Abcam, 1:1000), gp120, α-p24 and α-Nef were obtained from NIH AIDS Reagent Program, Manassas, VA, USA.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Concentration Assay, Incubation, Control, SDS Page, Comparison

Journal: STAR Protocols

Article Title: Protocol for an in vitro assay to study HIV-1 Tat methylation

doi: 10.1016/j.xpro.2023.102687

Figure Lengend Snippet:

Article Snippet: CDK9/cyclin T1, human, recombinant , Thermo Fisher Scientific , Cat# PV4131; Lot# 1860128B.

Techniques: Recombinant