Anti vapb

To validate knockout cell lines and cells treated with siRNAs, cells were lysed with IP lysis buffer (Thermo Fisher Scientific) supplemented with 1X protease inhibitor cocktail (Thermo Fisher Scientific) on ice for 30 mins. The cell lysates were centrifuged at 18,000 × g for 15 min at 4°C. The supernatant was collected and denatured in Laemmli buffer (with 10% beta-mercaptoethanol) at 95°C for 10 min. Proteins were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane (BIO-RAD, Hercules, CA, USA). The membrane was blocked with EveryBlot blocking buffer (BIO-RAD) for 1 h, and incubated with a primary antibody (1:1,000) overnight. The membrane was then washed with PBST for 10 min × 3 times and incubated with a corresponding secondary antibody (1:2,500). The membrane was then washed again with PBST for 10 min × 3 times before visualization of protein bands. Chemiluminescence was detected using ChemiDoc Imaging System (BIO-RAD). The following antibodies were used in this study: anti-Seipin antibody (Abnova Cat# H00026580-A02), anti-Plin 5 (Progen Cat# GP31), anti-VAPA (Sigma Cat# HPA009174), anti-VAPB (Sigma Cat# HPA013144), anti-Spastin (Sigma Cat# ABN368), and anti-GAPDH (Cell Signaling Cat# 2118).