M per protein extraction buffer

Automated Capillary Western was used to detect the target protein expression. After 6-day culture, GCs were washed using cold 1 × PBS and harvested by MPER protein extraction buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails. Protein samples were obtained by centrifugation at 12,000×g for 3 min at 4 °C, and the concentration of the protein supernatant was determined using a Micro BCA Protein Assay kit (23235;Thermo Scientific Pierce Biotechnology). Capillary electrophoresis were performed using the Wes system (ProteinSimple, Bio-Techne, USA) according to the manufacturer’s protocol. Protein samples were mixed with Wes sample master mix and heat denatured, then loaded onto the assay plate with other components which include primary antibodies (Supplementary Table 2), chemiluminescence substrate, blocking reagent, wash buffers, and secondary antibodies (anti-mouse DM-002 and anti-rabbit DM-001, ProteinSimple) in appropriate wells. The assay plate was loaded onto the Wes instrument for electrophoresis processing and analysis.

The protein samples for the Western blot analysis were prepared by lysing the cells using the M-PER protein extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) with 1X phosphatase inhibitor cocktail (PIC) (Cell Signaling Technology, Danvers, MA, USA) and 1X PMSF phenyl methylsulfonyl fluoride (Fluka, Buchs, Switzerland). The protein concentration was measured using the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific). The cell lysates were run in Bolt™ 4 to 12%, Bis-Tris, 1.0 mm, mini protein gel (Invitrogen, MA, USA) along with Spectra™ Multicolor Broad Range Protein Ladder (Invitrogen). They were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked in 5% milk, probed with the specific primary antibodies listed in Table 2, and finally in corresponding anti-rabbit or anti-mouse secondary antibodies. The protein bands were visualized using the chemiluminescence detection kit (Pierce, Thermo Fisher Scientific) and the images were captured in the ImageQuant™ LAS 4000 (Fujifilm, Tokyo, Japan). The densitometric analysis was performed using ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).

Lysosome purification was performed by magnetic fractionation with minor modifications [28 (link)]. In brief, HEK293 cells grown in 6-well culture plates were treated with GRN transcript-specific siRNA for 72 h. The culture medium was then replaced with medium containing dextran-coated magnetic nanoparticles (FluidMAG-DX; Chemicell) at a concentration of 0.5 mg/ml. After 6 h, cells were washed three times with PBS, fresh growth medium was added, and cells were cultured for an additional hour under normal cell culture condition. Cells were then washed twice in cold PBS and lysed in 400 μl hypotonic buffer (10 mM HEPES pH 7.4, 10 mM NaCl, 5 mM MgCl2 and protease inhibitor cocktails (1% w/v)) for 20 min on ice. The lysed cells were then sheared with a dounce homogenizer for 10 s in a microfuge tube, which was then placed in a microfuge tube magnetic separator. After 10 min, cell nuclei were removed and the magnetic fraction containing lysosomes was retained. The lysosomal fraction was washed twice with hypotonic buffer. The washed magnetic fraction was then lysed in M-Per protein extraction buffer (ThermoFisher Scientific) and normalized to protein amount by BCA assay before being analyzed by WB.