Anti hur antibody

WI-38 and NL20 cells were cultured in a chamber slide (Lab-Tek II, Rochester, NY, USA, Cat# 154526) and treated with 1µM niclosamide or DMSO for 48 h. Then, cells were fixed with 100% methanol (chilled at −20 °C) at room temperature for 5 min. Cells were then washed with ice-cold PBS and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Cat# T8787) for 10 min at room temperature. Cells were incubated with 1% bovine serum albumin (BSA, Fisher Scientific, Waltham, MA, USA, Cat# BP1605-100) and 22.52 mg/mL glycine (Sigma-Aldrich, Cat# G7126) in PBST (PBS with 0.1% Tween 20) for 30 min to block unspecific binding of the antibodies and then incubated with anti-HuR antibody (Santa Cruz, CA, USA, 3A2, Cat# sc-5621) at a 1∶200 dilution in 1% BSA overnight at 4 °C. Cells were then washed and incubated with anti-mouse IgG conjugated with FITC at a 1∶32 dilution in 1% BSA for one hour at room temperature. One μg/mL DAPI was then used for nuclear staining and was incubated for 1 min. Slides were mounted with SlowFade Gold antifade reagent (Invitrogen, Carlsbad, CA, USA, Cat# S36938) and DAPI containing mounting medium for fluorescence (Vector Laboratories, Newark, CA, USA, Cat# H-1200). Cells were imaged using an Olympus IX71 microscope using DP Controller and DP Manager software (3.3.1.222). Images were merged using ImageJ (1.53e).

The FFPE sections from ALD patients and normal controls were deparaffinized and boiled in citric buffer (pH = 6.0) for antigen retrieval. After blocking, the tissues were incubated with anti-NOX4 (1:1000, kindly provided by Dr. A. Shah, King’s College London, UK) at 4 °C overnight. The tissues were reacted with biotinylated secondary antibody followed by diaminobenzidine staining. For immunofluorescence, the slides were incubated with antibodies to NOX4 and αSMA (1:1000; Epitomics, Burlingame, CA) then probed with appropriate fluorescence conjugated secondary antibodies (Life Technologies, Carlsbad, CA). In vitro, after 100 μM acetaldehyde treatment for 24 hours, primary HSC were fixed with 4% paraformaldehyde, and then permeabilized with 0.2% TritonX-100 and 0.1% tween 20 in PBS. After blocking, the cells were probed with anti-HuR antibody (1:50, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), followed by appropriate fluorescence secondary antibody (Life Technologies) and mounted with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained by confocal microscopy.