Fusion solo 7s edge

Proteins were separated by 5–20% SDS-PAGE. Silver staining and GelCode Blue staining were performed using Silver Stain II Kit Wako (FUJIFILM) and GelCode Blue Safe Protein Stain (ThermoFisher Scientific), respectively, in accordance with the manufacturers’ protocols. For lectin and Western blotting, proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane, followed by blocking with TBS-T containing 1% BSA (for lectin blotting) or TBS-T containing 5% skim milk (for Western blotting). For lectin blotting, the membranes were incubated with HRP-conjugated lectin that had been diluted with 1% BSA in TBS-T. For Western blotting, the membranes were incubated with primary and HRP-conjugated secondary antibodies that had been diluted with 5% skim milk in TBS-T. Signals were detected with Western Lightning Plus-ECL (PerkinElmer) or SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific) using FUSION-SOLO 7 s EDGE (Vilber-Lourmat).

Proteins were separated in polyacrylamide gel and then transferred to polyvinylidene difluoride membranes (Millipore). These membranes were incubated with primary antibodies, followed by secondary antibodies conjugated to peroxidase. The proteins were visualized by enhanced chemiluminescence using Fusion SOLO.7S.EDGE (Vilber-Lourmat).

Cell lysates were prepared using the radioimmunoprecipitation (RIPA) buffer (Nacalai Tesque, Kyoto, Japan) or M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with the protease inhibitor cocktail (Nacalai Tesque). Western blot analysis was performed as described^{12 (link)}. Images were obtained with the GE Amersham Imager 600 (GE Healthcare, Milwaukee, WI, USA) or FUSION SOLO.7S.EDGE (Vilber-Lourmat, Marne la Vallée, France).

Cells were incubated with 100 nm TMR-conjugated HaloTag ligand (G8251, Promega) for 30 min. After being washed twice with PBS, cells were cultured in the starvation medium for the time indicated. Then, cells were harvested, and proteins were obtained. Proteins were separated by SDS-PAGE, and the gel was immediately visualized for in-gel fluorescence from TMR with a Fusion SOLO.7S.EDGE (Vilber-Lourmat). For the calculation of Halo–LC3 processing, the images of gels were captured with an exposure time of 30 s. The Halo–LC3 processing rate was calculated as the intensity of the free Halo band divided by the sum of the intensities of the free Halo and unprocessed Halo–LC3 bands. In the Halo–GFP processing assay, Halo processing is usually so low that the intensity of free Halo and unprocessed Halo–GFP bands cannot be visualized in the same image because the appropriate exposure time for each is different. Therefore, the images of gels were obtained twice with exposure times of 2 s and 10 min. The Halo–GFP processing rate was calculated as the intensity of the free Halo band in the 10-min exposed image divided by the intensity of the unprocessed Halo–GFP band in the 2-s exposed image (total Halo–GFP [processed + unprocessed] can be approximated as the intensity of unprocessed Halo–GFP in a 2 s exposed image, in which processed Halo can be hardly observed).

Cytokines secreted into culture medium were quantified using Mouse Cytokine Antibody Array (Membrane, 22 targets) (abcam, #ab133993) following the manufacturer’s protocol. Briefly, culture medium was substituted into fresh medium supplemented with 0.2% FBS prior to experiments, and culture medium was collected 8 h after the C12-iE-DAP stimulation (10 µg/mL). 100-fold diluted culture medium was incubated with an anti-cytokine antibody-arrayed membrane overnight at 4 °C. The target cytokines-trapped membrane was further incubated with the biotin-conjugated anti-cytokine antibody overnight at 4 °C, followed by the reaction with horseradish peroxidase (HRP)-streptavidin. The HRP-labelled cytokines was detected with chemiluminescence using a chemiluminescence imaging system FUSION SOLO.7S.EDGE (Vilber). Because the detection range in this kit is dependent on each cytokine, the detected membrane was adequately washed with TBS-T and iterated the above detection procedure for the undiluted culture medium.
The obtained images were adjusted by rolling ball background subtraction and quantified with using ImageJ/Fiji software. The quantified values were scaled with the values of negative controls and positive controls in each membrane.