Mms 101r

HeLa cells (American Type Culture Collection, ATCC, CCL-2) were transfected with vectors containing MAC-tagged gene of interest and cultured either with or without supplemental biotin. Bait proteins were detected with anti-HA antibody (Biolegend, MMS-101R, dilution 1:500), followed by Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, A-11001, 1:800). Biotinylated proteins were detected with Alexa Fluor 594 streptavidin (Thermo Fisher Scientific; S11227, 1:800). DAPI staining was used to determine the nuclei. Selected endogenous proteins were detected with specific antibodies (Supplementary Data 1a) and subsequently with Alexa Fluor 594 -conjugated anti-rabbit antibody (Thermo Fisher Scientific, A11012, dilution 1:800). Wide-field fluoresce microscope (Leica, Leica DM6000, Welzlar, Germany) with HCXPL APO 63×/1.40–0.60 oil objective was used to image the samples. For imaging sub-mitochondrial proteins, confocal microscopy (Leica TCS SP8 STED, Leica) with HC PL APO 93×/1.30 motCORR glycerol object was used. The image files were processed with LAS X (Leica), and ImageJ softwares.

Carbonate extraction was carried out as described previously (Yim et al., 2018 (link)), with the following modifications. Five OD₆₀₀ units of cells were harvested by centrifugation (2170 g, 5 min, 4°C), washed with distilled H₂O and subjected to lysis. The final lysate was subjected to centrifugation (20,000 g, 30 s, 4°C) to remove cell debris and transferred to a new pre-chilled tube. Centrifugation (20,000 g, 20 min, 4°C) followed after incubation with 0.1 M Na₂CO₃ (pH 11.5). Trichloroacetic acid (TCA)-precipitated ‘total’, ‘supernatant’ and ‘pellet’ fractions were centrifuged (20,000 g, 15 min, 4°C) and washed with acetone. Samples were resuspended in SDS sample buffer and analyzed by SDS–PAGE and western blotting. Mouse anti-HA primary antibody (1:10,000 dilution; MMS-101R, Biolegend) and horseradish peroxidase-conjugated anti-mouse secondary antibody (1:30,000 dilution; NCI1430KR, Thermo Fisher Scientific) were used for immunodetection.

Radiolabeled cell pellets were resuspended in 100 μl of lysis buffer [20 mM Tris-HCl (pH 7.5), 1% SDS, 1 mM DTT, 1 mM PMSF, and 1× Protease Inhibitor Cocktail (Quartett)] and mixed with 100 μl of ice-cold glass beads. Cell suspensions were vortexed for 2 min twice, keeping the samples on ice for 1 min in between. Subsequently, the samples were incubated at 60°C for 15 min and centrifuged (6000 g, 1 min, 4°C). Supernatant fractions were mixed with 500 μl of immunoprecipitation buffer [15 mM Tris-HCl (pH 7.5), 0.1% SDS, 1% Triton X-100, and 150 mM NaCl], 1 μl of anti-HA antibody (MMS-101R; Biolegend) and 20 μl of prewashed protein G–agarose beads (Thermo Fisher Scientific, Pierce) and rotated at room temperature for 3 h. The agarose beads were washed twice with immunoprecipitation buffer, once with ConA buffer [500 mM NaCl, 20 mM Tris-HCl (pH 7.5) and 1% Triton X-100], and once with buffer C [50 mM NaCl and 10 mM Tris-HCl (pH 7.5)]. The beads were incubated with 50 μl of SDS sample buffer [50 mM DTT, 50 mM Tris-HCl (pH 7.6), 5% SDS, 5% glycerol, 50 mM EDTA, 1 mM PMSF, 1× Protease Inhibitor Cocktail (Quartett) and Bromophenol Blue] at 60°C for 15 min, followed by Endo H (Promega) treatment at 37°C for 1 h. Protein samples were then loaded onto SDS–PAGE gels and separated by electrophoresis.

293T cells were transiently transfected with either HA or Flag-tagged USP11, and lysed in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 1 mM PMSF, 50 mM NaF, 10 mM Na₃VO₄, 1 μg/ml leupeptin and 1 μg/ml aprotinin) or in 0.1% NP40 lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 0.5 mM EDTA, 1 mM Na₃VO₄, 50 mM NaF, 1 mM β-glycerophosphate, 100 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin). Cell extracts were sonicated twice times followed by centrifugation to clarify lysate and incubated overnight with either anti-HA antibody (BioLegend MMS-101R) followed by capture with Protein G sepharose for 2 h, or Flag-M2-agarose affinity beads (Sigma; A2220). After ×4 washes with RIPA buffer, denatured proteins extracts were resolved by SDS-PAGE (NuPAGE Bis–Tris: ThermoFisher), left unfixed and stained with a MeOH-free ProtoBlue Safe Coomassie (S6-0044; Gene flow). Bands corresponding to the molecular weight of USP11 were excised and processed as below.

The mice were transcardially perfused with 0.9% saline (w/v), followed by perfusion with fresh fixative composed of 4% paraformaldehyde in a phosphate buffer (PB, 0.1 M, pH 7.4). The brains were collected and post-fixed overnight before coronal sections (50 μm thickness) were prepared using a vibratome (5100 mz Campden Instruments, Leicestershire, UK). After washing in PB several times, the sections were pre-incubated with 0.3% Triton X-100 (Sigma-Aldrich) in PB for 30 min at room temperature (RT) and incubated overnight with rabbit anti-GFAP antibody (1:1000 dilution, ab7260, Abcam, Cambridge, UK) or mouse anti-HA antibody (1:1000; MMS-101R, BioLegend, San Diego, CA, USA) at RT. Immunofluorescence was performed with secondary antibodies (Alexa Fluor 488-labeled anti-mouse antibody, 1:500; A11001, Invitrogen, Carlsbad, CA, USA or Alexa Fluor 594-labeled anti-rabbit antibody, 1:500; A21209, Invitrogen) for 2 h at RT. The sections were mounted onto glass slides and covered with coverslips using a drop of mounting medium (Dako North America, Carpinteria, CA, USA). The coverslips were sealed with nail polish to prevent the desiccation and movement of the samples under the microscope. Images were obtained using fluorescence microscopy (Axioplan2 Imaging, Carl Zeiss Microimaging, Oberkochen, Germany) and then subjected to analyses.