Tris buffered saline (tbs)

For Western blotting, the cells were lysed on ice in modified Radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L TrisHCl (pH 7.4), 50 mmol/L sodium fluoride, 150 mmol/L NaCl, 1% Nonident P40, 0.5 mol/L EDTA (pH 8.0)) supplemented with Complete Proteinase Inhibitor Cocktail (Roche, Mannheim, Germany). Supernatants of the samples were collected after centrifugation at 14,000× g and protein concentration was determined using a Bicinchoninic Acid Protein Assay Kit (ThermoFisher, Rockfold, IL, USA). Protein (20 μg of each sample) was electrophoresed on 4–12% Bis-Tris Gel (Life Technologies, Carlsbad, CA, USA) and transferred to the membrane. The membranes were blocked with 5% non-fat milk in 1× Tris-Buffered Saline (TBS) (Boston BioProducts, Ashland, MA, USA) for 1 h and probed with primary antibodies overnight. Membranes were washed with TBS and visualized using Super Signal West Pico chemiluminescent substrate (ThermoFisher). The following antibodies were used: anti-LDH-A (Abcam, Eugene, OR, USA) and anti-β-actin (Sigma Aldrich, Burlington, MA, USA).

Viral DNA detection (DNAscope) was performed by utilizing the sense probe targeting the reverse strand for each viral lineage of interest or cellular DNA utilizing a sense probe targeting the complementary CCR5 strand (Supplemental Table 2; data not shown). We modified the RNA-scope procedure by adding an RNase tissue pretreatment step (with ribonucleases A [25 μg/ml; Fisher Scientific] and T₁ [25 units/ml; Roche Diagnostics] in 1× Tris-buffered saline [TBS; Boston BioProducts] containing 0.05% Tween-20 [TBS-Tw] for 30 minutes at 37°C) following the RNA-scope pretreat three step, which was followed by a short denaturation step in which we incubated the slides at 60°C with warmed (60°C) sense probes for 10 to 15 minutes, and then immediately transferred the hybridization chamber to an oven set at 40°C and hybridized overnight (between 18 to 21 hours). Amplification and detection were performed as described for RNAscope previously, using 0.5X wash buffer for all washing steps. DNAscope validation was performed on 3D8 and ACH-2 cells diluted in uninfected CEM cells (generated as described previously) to make cell preparations at different concentrations ranging from 0% 3D8 or ACH2 cells to 100% 3D8 or ACH2 cells. Cell pellets were embedded in paraffin blocks, after which 4 to 6 μm sections were cut, and SIV DNA in situ hybridization was performed using DNAscope.