Nupage sample loading buffer

Protein samples were heated at 95 °C for 5 mins in NuPAGE sample loading buffer (Invitrogen) with DTT reducing agent (Invitogen) and run on a 4–12% Bis-Tris PAGE (Invitrogen) in 1xMES Buffer for 30mins at 200 V. Proteins were transferred to PVDF membranes using Transblot System (Biorad) set on a mixed size program for 7 mins. Membranes were blocked in 2%MILK-PBST for 1 h at room temp. Ten milliliter of HRP labelled anti-c-myc antibody (Miltenyi Biotech, diluted 1/5000 in block solution) was added to membranes for 1 h, then membranes were washed in PBST 3x5mins. Ten milliliter of ECL reagent (Invitrogen) was added to the membrane for 2 mins and chemiluminesence visualised using the Biorad Gel Doc System.

MDM were cultured as adherent monolayers in 6 well-plates at a density of 3×10⁶ cells/well and infected with HIV-1_ADA overnight. Following day, cells were washed with PBS and supplemented with fresh medium with or without CXCL8. LPS (2 ng/ml) was added to uninfected MDM. Three hours later, cells were collected by scraping in PBS. Cytoplasmic and nuclear protein extracts were isolated using nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific, Pittsburgh, PA). Equal amounts of protein samples (15 μg) were boiled with 4× NuPAGE loading sample buffer (Life Technologies) for 5–10 min, resolved by NuPage 4–12% Bis tris gel and subsequently transferred to a nitrocellulose membrane using i-Blot (Life Technologies). The membrane was incubated with primary antibody against NF-κB p65 (1∶1000, Cell Signaling) overnight at 4°C, washed and then incubated with anti-mouse or anti-rabbit goat antibody IgG conjugated to horseradish peroxidase (1∶10,000, Bio-Rad) for 2 h at room temperature. The membrane was then developed with SuperSignal west femto substrate (Fisher Scientific) in a Fluorochem HD2 Imager (ProteinSimple, Inc. Santa Clara, CA). GAPDH (1∶1000, Santa Cruz Biotechonology) and Lamin A/C (1∶1000, Cell Signaling Technology) were used as loading controls for cytoplasmic and nuclear extracts, respectively.

Equal amounts of protein samples (20 μg) were boiled with 4× NuPAGE loading sample buffer (Life Technologies) for 5–10 min, resolved by NuPAGE 4–12% Bis–tris gel and subsequently transferred to a nitrocellulose membrane using i-Blot (Life Technologies). The membrane was incubated with rabbit primary antibodies (cPLA₂, 1 : 500, Abcam; p-cPLA₂, 1 : 300, Abcam, CYP2E1, 1 : 500; Abcam and COX2, 1 : 500; Abcam) overnight at 4 °C, washed and then incubated with anti-mouse or anti-rabbit goat antibody IgG conjugated to horseradish peroxidase (1 : 1000, Bio-Rad) for 2 h at room temperature. The membrane was then developed with super-signal west femto substrate (Thermo, Rockford, IL, USA) in a Fluorochem HD2 Imager (proteinsimple, Inc., Santa Clara, CA, USA). GAPDH (1 : 5000, Cell Signaling) immunoblotting was used as a loading control.