Source data related to blots (CTNNBL1 restricts HIV-1 replication by suppressing viral DNA integration into the cell genome)

Published: 21 April 2022| Version 4 | DOI: 10.17632/ftj28s7v9j.4
Contributor:
Guoxin Liang

Description

Unprocessed data related to Fig.1,2,3,5,6,7 and Fig. S1,S3,S4,S5,S7 https://www.cell.com/cell-reports/fulltext/S2211-1247(22)00274-1

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IP and Western blotting Cells (5 x 106) were lysed with IP lysis buffer [50 mM Tris-HCl (pH 7.2), 50 mM NaCl, 1% NP- 40, 1 mM EDTA, 2% glycerol, and 1× Complete]. The lysates were incubated on ice for 30 min and centrifuged at 12,000 rpm for 10 min at 4°C. Supernatants were transferred to fresh tubes, and pellets were mixed with cold IP lysis buffer. Pellets were disrupted using sonication and centrifuged again at 12,000 rpm for 10 min at 4°C. Supernatants from the two extraction steps were pooled and incubated with the mixed protein A and G Dynabeads pretreated overnight with antibody at 4°C. As appropriate, cell lysates were incubated with 15 min for Benzonase digestion at room temperature, followed by inactivation with EDTA. IP products were washed with cold IP and PBST buffers 5 to 10 times (500 L/wash). Standard Western blotting was used to detect pull-down proteins. The antibodies used were rabbit polyclonal anti-CTNNBL1 (OriGene), rabbit polyclonal anti-LEDGF/p75 (Cell Signaling Technology), mouse monoclonal anti-HIV IN (Abcam), mouse monoclonal anti-HIV IN (Santa Cruz Biotechnology), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific), rabbit polyclonal anti--actin (Abcam), mouse monoclonal anti-HA (Thermo Fisher Scientific), mouse monoclonal anti-Myc (Santa Cruz Biotechnology), anti-rabbit and mouse IgG horseradish peroxidase (Abcam), rabbit and mouse IgG Trueblot (eBioscience), and rabbit or mouse IgG isotype control (Abcam).

Institutions

China Medical University

Categories

Western Blot

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