Immunobilon fl pvdf membrane

The total protein extract was prepared by homogenizing the cells in 1 × SDS sample buffer. All the protein samples were boiled for 5 min and then resolved in SDS-PAGE. The protein samples separated by SDS-PAGE were transferred to the Immunobilon-FL PVDF membrane (Merck Millipore, IPFL00010). After blocking with 5% non-fat milk in TBST (TBS, pH 7.4, 0.1% Tween-20), the membrane was incubated with primary antibody at 4 ℃ overnight. The membrane was washed and incubated with secondary antibodies in the dark for 2 h. The image was acquired by Li-Cor Odyssey Clx Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). The following primary and secondary antibodies were used for western blotting assay: SQSTM1 (Proteintech, 18420-1-AP), ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); Phospho-SQSTM1 (S403)-specific antibody (Cell Signaling Technology, 39786); FLAG tag (Prospec, ANT-146); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). Dylight 680, or Dylight 800-conjugated secondary antibodies (Thermo Fisher Scientific, A28183, A27042, 35518). See Additional file 1: Table S2 for further details and dilutions of all antibodies.