Nuclear factor κb nf κb p65

T0901317, bexarotene, ABC294640, SLM6031434, and FTY720 were purchased from Cayman Chemical (Ann Arbor, MI). Trichostatin A (#HY-15144) was purchased from MedChemExpress (Monmouth Junction, NJ). Synthetic Aβ_1-42 peptide (Peptide Institute, Osaka, Japan) was solubilized in hexafluoro-2-propanol at a concentration of 1 mg/ml to prevent self-aggregation. For immunoblotting and immunostaining, antibodies against human Aβ N terminal (clone 82E1; Immuno-Biological Laboratories, Gunma, Japan), ATP binding cassette transporter A1 (ABCA1) (clone HJ1; Abnova, Taipei, Taiwan), ApoE (#AB947; Merck Millipore, Burlington, MA), α-tubulin (clone 10G10; Fujifilm Wako, Osaka, Japan), β-actin (#5125; Cell Signaling Technology, Danvers, MA), GAPDH (#2118; Cell Signaling Technology), glial fibrillary acidic protein (GFAP) (#GTX108711; GeneTex, Irvine, CA), Lamin A/C (#10298-1-AP; Proteintech, Rosemont, IL), LXRα/β (#sc-377260; Santa Cruz Biotechnology, Dallas, TX), nuclear factor-κB (NF-κB) p65 (#8242; Cell Signaling Technology), RXRα (#sc-515929; Santa Cruz Biotechnology), SphK2 (#17096-1-AP; Proteintech), and 6His (#66005-1-Ig; Proteintech) were purchased from the indicated vendors.

For western blotting, all lysates were prepared in 2 × Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue (Bio-Rad, Hercules, CA, USA)). All cellular proteins were quantified using Bradford solution (Bio-Rad), according to the manufacturer’s instructions. The samples were then separated using SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Bio-Rad). After blocking with 4% (w/v) skim milk in Tris-buffered saline plus Tween (TBST, 25-mM Tris, 140-mM sodium chloride, and 0.05% (v/v) Tween^® 20), the membranes were incubated overnight with primary antibodies that were specific to the following target proteins: actin (MAB1501, 1:10,000 Millipore, MA, USA), IFT88 (13967-1-AP, 1:3000 Proteintech, Chicago, IL, USA), GLI2 (18989-1-AP, 1:3000 Proteintech), cleaved caspase-3 (#9661S, 1:2000 Cell Signaling Technology, MA, USA), phospho-NF-κB p65 (#3031, 1:1000 Cell Signaling Technology), and nuclear factor-κB (NF-κB) p65 (#8242, 1:2000 Cell Signaling Technology). For protein detection, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology).

Chondrocytes were isolated from the femoral condyle and tibial plateau of postnatal day 1 control and hTTR‐TG mice (n = 5, pooled), as described previously (Gosset et al., 2008). Chondrocytes were seeded on 6‐well plates (2 × 10⁵/well) and incubated for 24 h, and protein was extracted as described (Akasaki et al., 2015). Proteins were separated by NuPAGE (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose. The following antibodies were used: human TTR (Dako, Santa Clara, CA, USA), ADAMTS‐4 (Abcam, Cambridge, MA, USA), MMP‐13 (Abcam), nuclear factor‐κB (NF‐κB) p65 (Cell Signaling, Danvers, MA, USA), and phospho‐NF‐κB p65 (Ser536) (Cell Signaling). The primary antibodies were added and incubated at 4°C overnight. After secondary antibody application, signal detection was performed using an LI‐COR immunofluorescence detection system as described (Akasaki et al., 2014).