Ab137446

Cellular proteins were extracted using radioimmunoprecipitation assay protein extraction reagent (Beyotime Biotechnology Co., Shanghai, China) containing a mixture of protease inhibitors and phenylmethylsulfonyl fluoride (Roche, Basel, Switzerland). And 30 μg protein was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the protein was transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, United States), and blocked with 5% skim milk for 2 h. The membranes were incubated with specific antibodies at 4°C overnight, including rabbit anti-CD63 (1: 1000, ab134045, Abcam, Cambridge, United Kingdom), rabbit anti-CD9 (1: 1000, ab263019, Abcam), rabbit anti-GM130 (1: 1000, ab215966, Abcam), and rabbit anti-KPNA3 (1: 1000, ab137446, Abcam). GAPDH (1: 5000, ab181602, Abcam) was used as a control. Protein bands were visualized using the Millipore enhanced chemiluminescence Western Blotting Detection System (Millipore). The experiment was conducted in triplicates for three times independently.

The following primary and secondary antibodies were applied for immunodetection and immunostaining: anti-ataxin-3 (1:5000; 1H9, MAB5360, Merck, Darmstadt, Germany), anti-ataxin-3/C-terminal (1:2500; SA3637) [20 (link)], anti-KPNB1 (1:5000; H-300, sc-11367, Santa Cruz Biotechnology, Heidelberg, Germany), anti-KPNA3 (1:5000; ab137446, Abcam, Cambridge, United Kingdom), anti-CLPP (1:2500; 15,698–1-AP, Proteintech, St. Leon-Rot, Germany), anti-GAPDH (1:5000; 0411, sc-47724, Santa Cruz Biotechnology), anti-β-actin (1:5000; AC-15, A5441, Sigma-Aldrich), anti-vinculin (1:1000; E1E9V, 13,901, Cell Signaling Technologies, Frankfurt am Main, Germany), anti-lamin A/C (1:5000; 346, sc-7293, Santa Cruz Biotechnology), anti-GST (1:2500; B-14, sc-138, Santa Cruz Biotechnology), anti-α-spectrin (1:1000; AA6, Merck), anti-GFP (1:1000; B-2, sc-9996, Santa Cruz Biotechnology), anti-LC3 (1:1000; MBL-PD014, MBL International, Woburn, MA, US), anti-p62 (1:1000; 5114, Cell Signaling Technologies), and IRDye secondary antibodies goat anti-mouse 680LT, goat anti-mouse 800CW, goat anti-rabbit 680RD, goat anti-rabbit 800CW (all 1:10,000; LI-COR Biosciences, Bad Homburg, Germany), and Alexa Fluor 555 (1:500; Thermo Fisher Scientific).