Ab53841

For Western blot analysis, cells were lysed in lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, and protease inhibitors, pH 7.5) at 4°C for 30 min. Equal amounts of cell lysate (25 μg) were separated on 4–12% or 10% acrylamide gels (Thermo Fisher Scientific) and transferred onto 0.22 μm PVDF membranes (Millipore). After blocking with blocking buffer (TBST supplemented with 5% nonfat dry milk), membranes were probed with specific antibodies: mouse anti-CNP (ab6319, 1:2,000; Abcam), goat anti-HIV1 p24 (ab53841, 1:2,000; Abcam), rabbit anti-Tom20 (ab186735, 1:1,000; Abcam), rabbit anti-GAPDH (5174S, 1:1,000; Cell Signaling Technology), rabbit anti-Flag (14793S, 1:1,000; Cell Signaling Technology), and rabbit anti-β-tubulin (ab6046, 1:1,000; Abcam). Subsequently, blots were probed with species-specific IRdye secondary antibodies, and visualization was performed using an Odyssey infrared imaging system (Li-COR).

Western blot analysis was performed as previously described [35] (link), [52] (link) with minor modifications. Briefly, cultured cells in 60 mm dishes were scraped into lysis buffer (PBS plus protease inhibitors from Sigma). Thirty micrograms of protein was separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membrane (Bio-Rad). The blots were blocked in PBS-0.1% Tween-20 containing 5% nonfat milk and then incubated with antibodies at 4°C for 16 h. Primary antibodies were rabbit anti-human PGRN (Invitrogen 40–3400 at 1∶100) or goat anti-human PGRN (R&D Systems AF2420 at 1∶1000) and goat anti-HIV p24 (Abcam, ab53841 at 1∶1000). β-actin (Sigma-Aldrich, A2228) was used as the loading control. The secondary antibodies were HRP-conjugated anti rabbit or anti-goat IgG (Pierce/Thermo Scientific, at 1∶1,000) applied for 1 h at RT. Signals were developed using West Pico or Femto chemiluminescent reagents (Pierce/Thermo Scientific). Densitometry was performed using the NIH ImageJ software.

TZM‐bl cells exposed to HIV‐1 Gag‐mCherry viruses pseudotyped with JR‐FL Env (MOI 1) were fixed 3 dpi using 4% paraformaldehyde (PFA). Fixed cells were blocked/permeabilized using 10% fetal bovine serum (FBS), 0.5% saponin in PBS buffer (Immunofluorescence, IF, buffer). Primary antibodies recognizing HIV‐1 p24 (ab53841, Abcam) were diluted in IF buffer to 4 µg/ml and applied to cells for 1h in a humid chamber at 37°C. Anti‐goat secondary antibodies conjugated to Alexa 488 dye (A‐11055, Invitrogen) were diluted to 2 µg/ml in IF buffer and added to cells for 30 min at 37°C. Lipid droplet staining was performed adding Nile Red dye (Cat 60029, Biotium) diluted in PBS to 1 µg/ml, and the preparation was incubated for 10 min at room temperature. Excess of Nile Red dye was removed by washing twice with PBS before microscopy imaging.