Ab60178

Total protein was extracted from treated cells using 1% RIPA Lysis Buffer (Beyotime, Jiangsu, China) with a phosphorylation inhibitor (Roche, Indianapolis, USA). The total protein concentration for each sample was measured with a bicinchoninic acid (BCA) kit (Beyotime). Equal amounts of protein samples were then subjected to SDS-PAGE and proteins were then transferred to PVDF membranes (Beyotime). The membranes were blocked with 5% skim milk at room temperature for 1 hr and incubated with the primary antibodies against LOX-1 (1:1,000, ab60178, Abcam), NLRP3 (1:1,000, ab4207, Abcam), ASC (1:5,000, ab127537, Abcam), caspase1 (1:1,000, ab1872, Abcam), mature IL-1β (1:1,000, ab200478, Abcam) and β-actin (1:1,000, ab8226, Abcam). Subsequently, the membranes were incubated in anti-rabbit IgG horseradish peroxidase conjugated antibody (1:5,000; Abcam) for 1 hr at room temperature. The immune complexes were detected using the SuperSignal west pico kit (Shanghai solarbio Bioscience & Technology, China). The intensities of the signals were quantified using ImageJ software 6.0.

Total protein of ox-LDL-induced HAECs with transfection was extracted with radio-immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific). Next, total protein was segregated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8-12%, SDS-PAGE). Following that, the wet electrophoretic transfer method was executed for transference of the segregated protein to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Afterward, tris buffered saline tween (TBST) buffer with 5% skim milk was employed to block the PVDF membranes. After that, the PVDF membranes were incubated with primary antibodies overnight at 4°C. The antibodies were as follows: anti-Nod2 (ab31488, 1 : 1000, Abcam), anti-LOX-1 (ab60178, 1 : 1000, Abcam), and anti-β-actin (ab11003, 1 : 1000, Abcam). Then, the membranes were incubated with goat anti-mouse (ab205719, 1 : 2000, Abcam) or rabbit (ab205718, 1 : 2000, Abcam) IgG. β-actin was regarded as a loading control. Finally, the bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Mouse knee joints were used for histological evaluations. Firstly, 15% ethylenediaminetetraacetate (EDTA)-buffered saline solution was used to decalcify samples for 30 days. The samples were bathed in a graded series of ethanol solutions for dehydration and in xylene, and then embedded in paraffin blocks. They were then sectioned into 5 μm-thick slices. Paraffin-embedded samples were de-paraffinized in xylene, rehydrated using graded alcohol, and immersed in PBS. Slices were stained with H&E (Sigma-Aldrich, USA) for structural analysis of the bone matrix.
Immunohistochemistry was carried out according to a standard SABC protocol from the manufacturer. De-paraffinized slices were stained with rabbit antibodies against LOXs and goat anti-rabbit IgG/biotin (No. SP-0023; Bioss, Beijing, China). Rabbit monoclonal anti-LOX and anti-LOXL2 were purchased from Abcam (ab174316 and ab179810; Cambridge, MA, USA). Rabbit polyclonal anti-LOXL1 and anti-LOXL4 were purchased from Abcam (ab60178 and ab130646; Cambridge, MA, USA). Rabbit polyclonal anti-LOXL3 was purchased from Santa Cruz (sc-68939; Santa Cruz, CA, USA). The rehydrated samples were pretreated with 0.1% trypsin for antigen retrieval before staining. All stained slices were scanned with an inverted light microscope (Olympus-IX71, Japan) and analyzed via software (Aperio; Image Scope, USA).