Lc2001

Western blot analysis was performed to validate the release of fibrosis-related proteins and to elucidate the cell signaling pathways using α-SMA (ab7817, 1:1000), type-1 collagen (ab34710, 1:1000), total and phospho-SMAD (5678S, 8828S, 1:2000), total and phospho-JNK (sc7345, 9251S, 1:2000), total and phospho-ERK (sc514302, sc7383, 1:2000), and total and phospho-p38 (sc7149, 9211S, 1: 2000) antibodies. The treated cells were washed twice with PBS and lysed with lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L NaCl2, 0.5% Triton X-100, 1 mmol/L EDTA, 1% phosphatase inhibitor, 1% protease inhibitor (Sigma, St. Louis, MO)] at 30 min (SMAD, JNK, ERK, and p38) and 48 h (α-SMA and collagen) after treatments. The supernatant was collected after centrifugation at 12,000 rpm for 15 min and quantified using a BCA protein assay kit (Thermo Fisher Scientific). Protein (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (LC2001, Invitrogen, MA, USA). After blocking with 5% bovine serum albumin (BSA), the membrane was incubated with primary and secondary antibodies (HRP-anti-mouse; Thermo Fisher Scientific, HRP-anti-rabbit; AbFrontier, Korea, 1:5000). The signals were detected using an imaging system (iBright FL1000, Thermo Fisher Scientific) and quantified using the gel analysis plugin in ImageJ.⁴³ (link)

Cells collected on the day of HIV infection were lysed in RIPA buffer (50 mM Tris pH 8.0, 0.1% SDS, 1% Triton-X, 150 mM NaCl, 1% deoxycholic acid, 2 mM PMSF). Cell extracts were prepared in 4X LDS Buffer (Invitrogen #NP007) and 2X MES SDS Running Buffer (Invitrogen #NP002). After boiling at 100C for 5 min, 20 μg of the prepared samples were loaded into a NuPAGE 4–12% Bis-Tris Gel (Invitrogen #NP0321) and resolved. Following transfer onto a nitrocellulose membrane (Invitrogen #LC2001), blots were blocked in blocking buffer (PBS, 0.1% Tween, 5% milk) for 1 h, incubated with the MX2 primary antibody (Santa Cruz Biotech #sc-271527; 1:100) overnight, probed with secondary antibody (Cytiva #NA931, 1:1000) for 1 h, and visualized by chemiluminescence (Cytiva #RPN2232) on a Bio-Rad ChemiDoc Touch. Membranes were washed in PBST (PBS, 0.1% Tween) in between each step. After imaging the MX2 stain, membranes were stripped (ThemoFisher #46430), washed, probed with GAPDH:HRP (Bio-Rad #MCA4739P, 1:1000) for 1 h, and imaged with chemiluminescence.