Anti sqstm1

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.

Cells were washed in cold Dulbecco's phosphate-buffered saline (DPBS) and lysed in Modified Oncogene Science lysis buffer (MOSLB) (50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 μM Na₃VO₄, 10 mM HEPES, and 0.1%Triton X-100). Protein concentration was measured by either Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA; #500-0006) or BCA protein assay (Thermo Scientific, Rockford, IL, USA; 23227). SDS-PAGE was carried out with 20–40 μg of protein loading. Western blotting was performed according to standard protocol as previously described. The antibodies used in this study were anti-SQSTM1 (PROGEN Biotechnik GmbH, Heidelberg, Germany; GP62-C), anti-cytochrome c (Santa Cruz Biotechnology, H-104, sc-7159), anti-TOMM20 (Santa Cruz Biotechnology, FL-145, sc-11415), anti-NBR1 (Santa Cruz Biotechnology, sc-130380), anti-ACTB/β-actin (Sigma, Oakville, ON, Canada; A5316), anti-HA (Roche, Mississauga, ON, Canada; 11867423001), anti-PARK2 (Cell Signaling, Danvers, MA, USA; Prk8, #4211), anti-VDAC1 (Cell Signaling; #4866), and anti-MAVS (Cell Signaling, #8348), and anti-PINK1 (GeneTex, N3C3, Irvine, CA, USA; GTX107851). Protein levels were quantitated by densitometric analysis using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/) and normalized to those of ACTB.