Supernatants of the cell lysates in buffer A were used for immunoprecipitation of the purposed proteins as described elsewhere [16 (link),17 (link)]. The supernatants were mixed with protein G resin (GE Healthcare, Fairfield Easton, CT, USA) that had been absorbed with an antibody. The immunoprecipitates in supernatants of the cell lysate were denatured, subjected to denaturing polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes for immunoblotting.
For immunoprecipitation of the lysosome [18 (link)], we used buffer B (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF) and homogenized cell lysates with Potter-Elvehjem homogenizer. The homogenates were centrifuged at 150× g for 10 min in a tabletop centrifuge. The supernatants were gently mixed with an anti-SLC38A9 antibody-absorbed protein G resin according to their respective manufacturer’s instructions. The immunoprecipitates were denatured, subjected to denaturing gel electrophoresis, and blotted for immunoblotting.
For immunoprecipitation of the lysosome [18 (link)], we used buffer B (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF) and homogenized cell lysates with Potter-Elvehjem homogenizer. The homogenates were centrifuged at 150× g for 10 min in a tabletop centrifuge. The supernatants were gently mixed with an anti-SLC38A9 antibody-absorbed protein G resin according to their respective manufacturer’s instructions. The immunoprecipitates were denatured, subjected to denaturing gel electrophoresis, and blotted for immunoblotting.
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