Journal: Mobile DNA
Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Figure Lengend Snippet: SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
Article Snippet: Immunoprecipitation assays 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies).
Techniques: Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Software, Amplification, Agarose Gel Electrophoresis, Immunoprecipitation