Anti tfeb

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.

For TFEB and TFE3 immunofluorescence, cells treated with mitochondrial stressors were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Cells were then blocked with 10% bovine serum albumin in PBS. After sequential incubation of cells with anti-TFEB (Bethyl Laboratories, 1:200) or anti-TFE3 Ab (Sigma, 1:200) at 4 °C overnight and then with anti-rabbit secondary Abs conjugated to Alexa Fluor 488 (Invitrogen) for 1 h, fluorescence was visualized with an LSM780 confocal microscope (Zeiss).

Anti-GAPDH (6C5), anti-GFP (IF), anti-SM22α, and anti-TBX18 were purchased from Abcam. Anti-CD31 was purchased from BD Pharmingen. Anti-TFEB (WB) was purchased from Bethyl Laboratories. Anti-Cre Recombinase (D7L7L), anti-Flag tag, anti-PDGFRα (D1E1E), anti-PDGFRβ (28E1), anti-Slug (C19G7), Anti-TFEB (ChIP-seq), and anti-Vimentin (D21H3) were purchased from Cell Signaling Technology. Anti-TGIF1 (H-172) and anti-WT1 (C19) were purchased from Santa Cruz Biotechnology. Anti-αSMA (1A4) and anti-vinculin (V9131) were purchased from Sigma-Aldrich. Anti-GFP (IHC), anti-cTnT, and anti-ZO1 were purchased from Thermo Fisher Scientific. HRP goat anti-mouse and goat anti-rabbit secondary antibodies (WB) were purchased from Jackson ImmunoResearch Laboratories. EnVision+ System- HRP Labelled Polymer Anti-Rabbit (IHC) was purchased from Dako. Alexa Fluor 555 donkey anti-mouse, Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 647 goat anti-rat and Alexa Fluor 488 goat anti-chicken secondary antibodies (IF) were purchased from Thermo Fisher Scientific. Catalog numbers, dilutions and validation information for the antibodies is reported in Supplementary Table 1.

For immunofluorescence analysis, cells were fixed in 4% (Vol/Vol) paraformaldehyde for 15 min then blocked in 8% (Vol/Vol) HyClone Fetal Bovine Serum (Thermo Scientific) for 1 h at room temperature. This was followed by incubation with primary antibodies in blocking buffer containing 0.2% Saponin (Sigma-Aldrich) over night at 4°C. Cells were then washed with PBS and incubated with the corresponding fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594, Invitrogen) at 1:500 for 1 h at room temperature. Cells were mounted in Vectashield mounting medium containing DAPI (VectorLabs) to label the nuclei. The following primary antibodies were used (all at 1:200 dilution unless otherwise stated): anti-LMX1 (Millipore), anti-Nurr1 (R&D Systems), anti-VMAT2 (Proteintech, 20873-1-AP), anti-GIRK2 (Proteintech, 21647-1-AP and Millipore, AB5200), anti-MAP2 (Millipore, MAB3418), anti-FoxA2 (Millipore, AB4125), anti-mTOR (Cell Signaling, 2972) 1:100, anti-phospho-mTOR-Ser2448 (Cell Signaling, 5536) 1:100, anti-S6 Ribosomal Protein (Cell Signaling, 2217) 1: 200, anti-phosphoS6-Ser235/236 (Cell Signaling, 2211 and 4856) anti-LAMP1 (U. Iowa Developmental Hybridoma Bank, H4A3) 1:100, anti-Tuj1 (Neuromics, MO15013 and BioLegend, 801207), anti-TFEB (Bethyl Laboratories, A303-673A), anti-Tyrosine Hydroxylase (Sigma-Aldirch, T1299), and anti-Bip/Grp78 (abcam, ab21685).

Western blot analysis was performed as previously described.²³ The membranes were incubated at 4°C overnight with the following primary antibodies: anti‐collagen I (Abcam, ab34710), anti‐TGF‐β1 (Abcam, ab92486), anti‐fibronectin (Abcam, ab23750), anti‐LC3B (Sigma, L7543), anti‐p62/SQSTM1 (Abcam, ab91526), anti‐Cathepsin B (Abcam, ab58802), anti‐phospho‐mTOR (Ser2448) (Cell Signaling, 5536S), anti‐mTOR (Cell Signaling, 2983S), anti‐phospho‐4E‐BP1(Thr37/46) (Cell Signaling, 2855S), anti‐4E‐BP1 (Cell Signaling, 9644S), anti‐phospho‐TFEB (Ser142) (Millipore, ABE1971‐I), anti‐TFEB (BETHYL, A303‐672). β‐actin (Santa Cruz, sc‐47778) and GAPDH (Absin Bioscience, abs132004) were used as the loading control in vivo and in vitro experiments, respectively. The membranes were washed with PBS‐T five times and then incubated with horseradish peroxidase‐labelled secondary antibody (Beyotime Biotechnology, China) for 1 h at room temperature. After visualized with Clarity Max™ Western ECL Blotting Substrates (Bio‐Rad, Hercules, CA, USA), the Azure C500 Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA) was used to detect the immunoreactive signals. The optical density of each band was then quantified using ImageJ software (NIH).

Cells were lysed in ODG buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 2% octyl-β-D-glucoside, 5% glycerol, 1 mM Na₃VO₄, 10 mM NaF, and protease inhibitors). Equal amounts of total proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked and incubated with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Signals were visualized on a WSE6200H LuminoGraph II (ATTO, Tokyo, Japan). Representative blots obtained from at least three independent experiments are shown. For pull-down assays in E. coli, cell lysates expressing His-tagged p18 were incubated with HisTrap beads, and precipitates were subjected to Coomassie Brilliant Blue staining and western blotting. The following antibodies were used: anti-p18, anti-p14, anti-MP1, anti-HBXIP, anti-p10, anti-mTOR, and anti-phospho-S6K (Cell Signaling Technology), anti-actin, anti-LAMP1 and anti-S6K (Santa Cruz Biotechnology), and anti-TFEB (Bethyl Laboratories Inc). Catolog numbers and dilutions of these antibodies are listed in Supplementary Table 5. All experiments were repeated at least three times, and representative blots are displayed in all figures. Uncropped images of western blots are shown in Supplementary Figs. 17–20.