Mouse anti gapdh clone 6c5

Liver and spleen samples were homogenized in radioimmunoprecipitation assay buffer containing proteinase and phosphatase inhibitors (Roche, Mannheim, Germany). Lysates were centrifuged at 15,000× g, 4 °C for 15 min to remove insoluble material, and the supernatant was collected. The protein concentration was measured using a Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Typically, 100 µg of protein was loaded for electrophoresis on a 4–15% precast Tris-glycine gradient gel (BioRad, Munich, Germany). The protein was subsequently transferred to a nitrocellulose membrane using semi-dry transfer apparatus (BioRad, Munich, Germany) for immunodetection analysis. The target proteins were detected by rat anti-mouse F4/80 Cl:A3-1 (1:500, Biolegend, San Diego CA, USA), rat anti-CD68 clone FA-11 (1:200, Bio-Rad Laboratories, Raleigh, NC, USA), anti-cathepsin D antibody clone EPR3057Y (1:2,000 Abcam, Cambridge, UK), and mouse anti-GAPDH clone 6C5 (1:10,000, Abcam), each in tris-buffered saline and tween supplemented with 5% skimmed milk powder (Sigma Aldrich, Munich, Germany). Fluorescent conjugate secondary antibodies were applied for the detection with a Li-Cor Odyssey imaging system (Bad Homburg, Germany).

Cellular fractionation was completed as previously described [52 (link)]. Briefly, at 24 hrs post transfection, HEK293T cells were washed once with PBS and collected in 500 μL homogenization buffer (150 mM KCl, 20 mM HEPES pH 7.4, 2 mM EDTA) containing protease inhibitors and passed 30 times through a 27G-needle. The lysate was centrifuged at 2000 x g for 5 min at 4°C to remove the nuclear fraction. The supernatant was subsequently centrifuged at 100,000 x g for 1 h at 4°C to pellet the membrane fraction. The supernatant was removed (cytoplasmic fraction) and the pellet (membrane fraction) was resuspended in 80 μL of homogenization buffer. Twenty micrograms of cytoplasmic and membrane fractions were separated by SDS-PAGE and blotted with anti-GFP rabbit serum (Life Technologies). Rabbit anti-calnexin [clone ab13505] and mouse anti-GAPDH [clone 6C5] antibodies (Abcam) were used as markers of the membrane and cytosol fractions, respectively.