Supersep ace precast gels

Western blotting was carried out as described previously^{55 (link)}. In brief, cells were washed with cold PBS 3 times to completely remove nanoparticles, and then harvested and lysed in RIPA buffer (9806, CST) containing protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1% phosphatase inhibitor (P5726, Sigma-Aldrich). The lysates were centrifuged at 14000 rpm for 30 min at 4 °C. The protein concentration was measured using a Bradford protein assay kit (T9310A, Takara, Shiga, Japan). Protein samples were then mixed with a 2 × Laemmli sample buffer (161–0737, Bio-Rad, CA, USA) and heated at 95 °C for 5 min. Equal amounts of protein were then applied to 5–20% SuperSep® Ace precast gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and transferred to Immun-Blot PVDF membranes (1620174, Bio-rad). The membranes were blocked with 5% skim milk buffer for 1 h at room temperature, and then incubated overnight at 4 °C with the following ab: anti-ACTB (1:5000), anti-MAP1LC3B (1:1000), and anti-p62/SQSTM1 (1:2000). Lastly, they were incubated for 1 h with HRP-conjugated anti-mouse or anti-rabbit ab and visualized with an enhanced chemiluminescence detection system (32106, PIERCE, Rockford, IL, USA).

The protein solutions were mixed with SDS-PAGE sample buffer (final concentration of components: 2% SDS, 10% (w/v) glycerol, 0.002% bromophenol blue, 50 mM Tris-HCl, pH 6.8) to prepare the samples for SDS-PAGE analysis. To denature the proteins, the mixed solution was heated at 95 °C for 5 min. Samples were subjected to SDS-PAGE using SuperSep Ace precast gels (Wako Pure Chemical Industries, Japan) and the running buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). 15% gels were used unless otherwise indicated. Visualization of fluorescent bands and the image capturing were carried out on a ChemiDoc XRS + Imaging System (BIO-RAD) equipped with a bandpass filter for GFP detection (520 nm, full-width half-maximum [FWHM] = 20 nm, Bio-Rad) under UV illumination. For in-gel fluorescent detection, BenchMark™ Fluorescent Protein Standard (ThermoFisher, product # LC5928) was used as marker proteins. After detection of the fluorescent bands, the gels were stained with CBB Stain One (Nacalai Tesque). Gel image was captured using a ChemiDoc XRS + Imaging System.