Sample buffer

Cells were lysed and sonicated in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (cOmplete cocktail EDTA-free, Roche). Protein extracts were boiled in sample buffer (BioRad), separated by SDS–PAGE under reducing conditions and transferred to nitrocellulose filters (BioRad) by semi-dry electro-blotting. Nuclear fractions for TIP5 were obtained using NE-PER cell fractionation kit (Thermo Scientific, #78833). Primary antibodies are listed in Supplementary Table 1. Immunoreactive bands were visualized by chemi-luminescence (BioRad) and a BioRad ChemiDoc XRS imaging system. Each experiment was performed on 3 biological replicates.

Whole cell extracts and immunoblotting were performed as described earlier (8 ). Soluble proteins were prepared by centrifugation of cell lysates for 15 min in a microfuge at 4 °C, 18,000g. To assay insoluble proteins, remaining cell pellets were resuspended in solubilization buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl, 2% SDS, 2 mM DTT, 1% Triton X-100, 8 M urea, and 1× protease inhibitor cocktail) and incubated at room temperature for 5 min, followed by centrifugation at 18,000g for 15 min at 4 °C in a microfuge. The supernatant was then heated with in sample buffer (Bio-Rad, Cat. No. 1610737) for 5 min at 37 °C. Antibodies recognizing the following epitopes were used for immunoblotting at the indicated dilutions: Acss2, 1:1000 (Cell Signaling Technology, Cat. No. 3658); GFP, 1:1000 (Invitrogen, Cat. No. A11121); α-tubulin, 1:5000 (Sigma, Cat. No. T9026); V5, 1:1000 (Invitrogen, Cat. No. R96025); LC3B, 1:1000 (Cell Signaling Technology, Cat. No. 2775); ubiquitin, 1:1000 (Cell Signaling Technology, Cat. No. 3936); HRP-linked anti-mouse IgG (Cell Signaling Technology, Cat. No. 7076); HRP-linked anti-rabbit IgG (Cell Signaling Technology, Cat. No. 7074).

After iPS-MLs or SH-SY5Y cells were cultured with the appropriate TTRs for 3 days, culture supernatants were mixed with sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) containing 2-mercoptoetanol, and boiled at 95°C for 5 min. Resultant samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). Polyclonal rabbit anti-human TTR antibody (Dako; diluted 1:1000) was used as the primary antibody, and polyclonal goat anti-rabbit antibody conjugated with HRP (Dako; diluted 1:5000) as the secondary antibody. The reaction was visualized using ECL prime reagents (GE Healthcare, WI, USA), and detected using a LAS-4000EPUVmini (GE Healthcare).

Western blotting was performed to measure protein changes. In brief, the samples (tissue and cell samples) were lysed in RIPA buffer (P0013C, Beyotime, Guangzhou, China) and mixed with sample buffer (No. 1610737, Bio–Rad) at a 1:1 ratio. Equal amounts of protein samples were separated by 10% SDS–PAGE and blotted onto PVDF membranes after transmembrane electrophoresis. The antibodies used included Runx1 (No. 39000, ChIP grade, Active motif, Carlsbad, CA) and FLAG (ab125243, Abcam, Cambridge, UK and 9A3, Cell Signaling Technology, Boston, MA, 1:1 000 dilution). The secondary antibodies included mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Biotech, Delaware Avenue, CA, 1:2 000 dilution) and m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotech, 1:2 000 dilution).

In preparation for Western blot analysis, supernatants and lysates were diluted in sample buffer (Bio-Rad) containing 1% 2-mercaptoethanol and heated at 80 °C for 5 min. Samples were resolved on a 10% acrylamide gel and proteins were transferred to a nitrocellulose membrane. Specific proteins were detected using anti-IL-1α, anti-IL-1β (both 0.1 μg/ml), or anti-ubiquitin antibodies (0.2 μg/ml). Subsequently, blots were incubated with either HRP-labeled anti-IgG antibody (for IL-1α or IL-1β; 0.25 μg/ml) or HRP-labeled anti-light chain IgG antibody (ubiquitin; 0.25 μg/ml). Proteins were visualized using enhanced chemiluminescence reagents (Thermo Scientific, Waltham, MA).

Protein samples were prepared by mixing one part of sample with one part of Bio-Rad Sample Buffer and then boiled at 100 °C for 5 min. Proteins were separated in 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane at 200 mA for 2 h in ice treatment. The blot was blocked with 5% fat-free dry milk suspended in 1× TBST for 15 min at RT. Resulting blot was incubated with antibodies (1:500–1:1000), including connexin43/β-catenin/active β-catenin/lef-1 for overnight at 4 °C, followed by incubation with 1:2000 anti-IgG-HRP (AlexSeries, Abcam) for 2 h at RT. Signals from the blots were obtained using a Western Blotting Luminol Reagent Kit.