Anti ctsd

For staining of cells, INS-1E were plated onto coverslips were fixed with 4% paraformaldehyde and immunostaining performed as in [18 (link)]. The primary antibodies used in this study included anti-CTSD (Santa Cruz Biotechnologies, SC-6487), anti-NFATC1 (BD Pharmingen, 556602) and anti-SQSTM1 (Progen, GP62-C). Cells were imaged using a Nikon TE2000 (×100). Lysosomal puncta as determined by CTSD puncta were quantified using Blobfinder software (Uppsala University, Sweden). TFEB and NFATC1 translocation were quantified by measurement of the mean pixel intensity for the nuclear and cytoplasmic compartments using ImageJ and the nuclear:cytoplasmic ratio calculated. For these calculations, >5 images were taken per condition for each independent experiment.
For immunostaining of tissue, indirect immunofluorescence staining was carried out as in [18 (link)]. The primary antibodies used in this study included anti-CTSD (Proteintech, 21327-1-AP), anti-INS (Dako, A0564), anti-SQSTM1 and anti-TFEB (Bethyl Laboratories, A303). Tissue was imaged using a Nikon Eclipse TE2000-S and 10–15 islets imaged per condition. The intensity of INS staining and SQSTM1, TFEB and CTSD puncta within INS positive areas was quantified using FIJI software.

Materials were purchased as follows: human recombinant IL-2 (Imunase 35) (Shionogi Pharmaceutical, Osaka, Japan); C₆-NBD ceramide, C₆-NBD sphingomyelin, and C₂-ceramide (Matreya, Pleasant Gap, PA, USA); CTSB specific inhibitor IV (CA-074 Me) (Merck Millipore, Darmstadt, Germany); pepstatin A and desipramine (Sigma-Aldrich, St. Louis, MO, USA); anti-caspase-8, anti-caspase-9, anti-PARP, antilysosomal-associated membrane protein 1 (Lamp1), anti-Lamp2, anti-ASM, anti-CTSB, anti-CTSD, and anti-β-actin antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); Annexin V-conjugated FITC and monoclonal antibody against XIAP (BD Biosciences, Beverly, MA, USA); antiactive caspase-3 (p17) antibody and horseradish peroxidase–conjugated secondary antibodies (Promega, Madison, WI, USA); anti-caspase-3, anti-pan-cadherin, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (Cell signaling, Danvers, MA, USA); and anti-ceramide monoclonal IgM Ab clone (NHCER-2) was produced previously in our lab.^{65 (link)} Alexa Fluor-conjugated (AF-conjugated) secondary antibodies were from Molecular Probes (Eugene, OR, USA). 4',6-Diamidino-2-phenylindole (DAPI) was obtained from Roche Applied Science (Penzberg, Germany).