Effects of Retinoic Acid on mTOR, SAMHD1 and TCF4 Expression in Macrophages

Description

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 www.BioRxiv.org)

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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.

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