Retinoic Acid Enhances HIV-1 Reverse Transcription and Transcription in Macrophages via mTOR-Modulated Mechanisms

Published: 5 June 2024| Version 1 | DOI: 10.17632/5b38g36z9v.1
Petronela Ancuta


Monocyte-derived macrophages (MDMs) were generated from monocytes of HIV-uninfected individuals in the presence or the absence of all trans retinoic acid (ATRA). MDMs were tested by western blotting for the expression of mTOR, S6K, and SAMHD1 protein expression/phosphorylation, as well as TCF4 protein expression. This file is linked to the manuscript entitled "Retinoic Acid Boosts HIV-1 Replication in Macrophages via CCR5/SAMHD1-Dependent and mTOR-Modulated Mechanisms" by Jonathan Dias, Petronela Ancuta et al., submitted for publication October 5th, 2023 (reprint available on


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Western Blotting Methods. Total lysates of MDMs (3x106 per condition) were generated using the Radio-immunoprecipitation Assay (RIPA) Buffer 1X (Cell Signaling, Danvers/United States) containing phosphatase (PhosSTOP; Roche, Basel/Switzerland) and protease inhibitors (Complete, Mini, EDTA-free protease inhibitor; Roche, Basel/Switzerland). Total protein content from each condition was quantified using DC Protein Assay (Bio-Rad, Hercules/United States) in triplicate samples. SDS-PAGE gel electrophoresis was performed on a gradient polyacrylamide gels for 75 minutes at 130 volts and transferred on immobilon-PSQ polyvinylidene difluoride (PVDF) membranes (Sigma, St. Louis/United States) for 75 minutes at 100 volts. PVDF membranes were blocked with Tris-Buffered Saline (TBS) 0.1% Tween 5% Bovine Serum Albumin (BSA) for 45 minutes and incubated overnight with primary antibodies against target proteins at 4 oC (Supplemental Table 1). PVDF membranes were washed four times with TBS 0.1% Tween and incubated with HRP-linked secondary Abs (Supplemental Table 1) for one hour at room temperature. All Abs were diluted with blocking TBS 0.1% Tween 5% BSA. PVDF membranes were washed four times and proteins were revealed with chemiluminescence western blotting substrates (Bio-Rad, Hercules/United States). PVDF membranes were reused by using a reblot stripping solution (Sigma St. Louis/United States). A Chemidoc imaging system from Bio-Rad was used for chemiluminescence and colorimetric detection to visualize the bands on PVDF membranes and Image lab software (Sigma St. Louis/United States) was used to quantify the band intensity between each condition and each donor.


Centre de recherche du CHUM, Universite de Montreal


Mammalian Target of Rapamycin, Western Blot, Retinoic Acid


Canadian Institutes of Health Research

PJT #178127

Canadian Institutes of Health Research


Canadian Institutes of Health Research


Canadian Institutes of Health Research

PJT #153052