Phospho sqstm1 s403 specific antibody

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.

Immunostaining was performed as described previously [12 (link)]. In brief, after treatment, cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized for 15 min with 0.2% Triton X-100 (Merck Millipore, 94101-L), and then blocked with 5% goat serum for 1 h. they were subsequently incubated with primary antibodies overnight at 4 °C followed by incubation for 1 h with secondary antibodies at room temperature. Their nuclei were stained with a DAPI solution. Images were captured with a fluorescent microscope (Nikon Eclipse 80i equipped with Nikon PLAN FLUOR × 40 objective) or Nikon confocal microscope (Nikon, N-STORM and A1). Photographic images were resized and analyzed by ImageJ software. The following primary antibodies were used for immunostaining: SQSTM1 (Santa Cruz Biotechnology, sc-28359); K48-linked Ub chain-specific antibody (Merck Millipore, 05-1307); FLAG Tag (Prospec, ANT-146); Phospho-SQSTM1 (S403)-specific antibody (Cell Signaling Technology, 39786), Alexa Flour 488- or 568-conjugated secondary antibodies (Thermo Fisher Scientific, A11034, A11029, A11031, A11036); See Additional file 1: Table S2 for further details and dilutions of all antibodies.