Western blot analysis was performed as previously described (92 (link)) using primary antibodies and secondary Horseradish peroxidase-coupled antibodies listed in Table S3 . Chemiluminescence was detected on a Chemidoc Touch Imaging System (Bio-Rad). Data was processed with ImageLab (Bio-Rad) and normalized to total protein (stain-free detection) (94 (link)).
For the compound stimulation cells were grown in 6-well plates and treated as indicated (24 h) with chloroquine (Sigma-Aldrich, #C6628) or EDME. For Rab11-overexpression cells were transiently transfected with pEGFP-C1-Rab11-WT (Addgene, #12674) or pmaxGFP-plasmid (Lonza, #VCA-1001) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, #100022057) according to manufacturer’s instructions and incubated (48 h). For cathepsin B release cells were grown overnight, medium was exchanged to FCS-free medium, and treated with ionomycin (5 μM) (Sigma, #10634) for 10 min (95 (link)). Supernatant was concentrated using Merck Amicon centrifugal units (Thermo Fisher Scientific, #10341782).
For the compound stimulation cells were grown in 6-well plates and treated as indicated (24 h) with chloroquine (Sigma-Aldrich, #C6628) or EDME. For Rab11-overexpression cells were transiently transfected with pEGFP-C1-Rab11-WT (Addgene, #12674) or pmaxGFP-plasmid (Lonza, #VCA-1001) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, #100022057) according to manufacturer’s instructions and incubated (48 h). For cathepsin B release cells were grown overnight, medium was exchanged to FCS-free medium, and treated with ionomycin (5 μM) (Sigma, #10634) for 10 min (95 (link)). Supernatant was concentrated using Merck Amicon centrifugal units (Thermo Fisher Scientific, #10341782).
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