To pro 3 iodide

Reagents were obtained from the following sources: antibodies to phospho-4E-BP1 (Thr37/46) from Cell Signalling (#2855); H3 antibodies from Abcam (#ab1791); anti-rabbit IgG secondary antibodies from KPL (#074–1506); Lambda Protein Phosphatase (Lambda PP) from New England Biolabs (#P0753S); ARTseqTM kit from Epicentre (#RPHMR12126); RNaqueous kit from Ambion (#AM1912); Torin 1 from Tocris (#4247); protease inhibitor cocktail from Sigma-Aldrich (P2714-1BTL); Halt™ Phosphatase Inhibitor Cocktail from Thermo Scientific (#78420); precast gels from BioRad (#4566033 and #4565013); To-Pro-3 iodide from Molecular Probes (#T3605); VECTASHIELD® from Vector Laboratories (#H1000); EdU from Thermo Fisher Catalog (#A10044); Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit from Thermo Fisher (#C10428); TMG-cap antibodies from Santa Cruz (#sc-32,724); Zymo RNA Clean & Concentrator-25 kit (#R1018); Ribo-Zero (#MRZG12324); Clarity Western ECL Substrate from BioRad (#170–5060).

Anti-SNAP-25, anti-actin, anti-Flag, anti-GST, anti-syntaxin, peroxidase conjugated anti-rabbit IgG and anti-chicken IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-SNAP-25^pThr138 (Abgent, San Diego, CA, USA), anti-MYPT1^1-296 [20 (link)], anti-PP1cδ (Millipore, Billerica, MA, USA), anti-GAPDH (Santa Cruz, CA, USA), anti-CPI17^pThr38 [22 (link)], horseradish peroxidase-linked anti-mouse IgG (Cell Signalling, Danvers, MA, USA), Clean-Blot IP Detection Reagent (Thermo Scientific, Waltham, MA, USA) and Texas Red-X phalloidin (Life Technologies, Carlsbad, CA, USA) were purchased as indicated. Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 546-conjugated anti-mouse IgG, Alexa Fluor 546-conjugated anti-goat IgG and To-Pro-3 iodide were obtained from Molecular Probes (Eugene, OR, USA).

The hemolymph was collected from control and WSSV-injected shrimps at 6, 24 and 48 hpi, as well as from moribund shrimps, and immediately fixed by incubation in 4% (w/v) paraformaldehyde at room temperature for 10 min. The fixed hemocytes were washed in PBS (centrifugation stage at 800×g at 4°C for 10 min) and resuspended in PBS. About 10⁶ hemocytes were attached onto each SuperFrost microscope slide by centrifugation at 1000×g for 10 min. Slides were blocked in 10% (v/v) fetal bovine serum in PBS at room temperature for 1 h and then probed with purified rabbit polyclonal antibody specific to PmVRP15 and purified mouse monoclonal antibody specific to VP28 (WSSV capsid protein) for 1 h at room temperature and washed three times in 0.05% (v/v) Tween-20 in PBS to remove non-specific binding. The Alexa 488-conjugated goat anti-rabbit IgG and Alexa 568-conjugated goat anti-mouse IgG secondary antibodies (Invitrogen) were applied to the slides and incubated at room temperature for 1 h. The slides were then washed three times as above, incubated with TO-PRO-3 iodide (Molecular Probes) to stain the nuclear DNA and then washed once with PBS. Mounting medium, ProLong Gold antifade (Molecular Probes), was applied and the slides were examined by CLSM (Olympus). Bright field and fluorescence images were collected for the analyses.

For immunocytochemistry, cells grown on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing step with PBS, cells were incubated in a blocking solution (2% goat serum in PBS) and then incubated with primary antibodies. For immunohistochemistry, mouse brain sections were prepared according to the method previously described24 (link). The following primary antibodies were used: anti-p62 (CST), anti-ubiquitin (Santa Cruz), anti-αS (CST), anti-CHOP (Santa Cruz), anti-phospho-neurofilament (Covance), anti-phospho-c-Jun (CST), and anti-cleaved caspase-3 (CST). Positive immunostaining was detected using Alexa 488- and Alexa 647-conjugated secondary antibodies (Molecular Probes). Nuclei were counterstained with DRAQ7 (BioStatus) or TO-PRO3 iodide (Molecular Probes). Fluorescence images were analyzed with an FV300 confocal laser scanning microscope (Olympus).

RPE monolayers (ARPE-19 and ARPE-19 + shDJ-1) transduced with the different adenovirus constructs mentioned above and stimulated with 0 and 200 μM H₂O₂ were fixed in paraformaldehyde (4%) made in PBS for 20 min at room temperature (RT), followed by quenching in 50 mM ammonium chloride made in PBS for 20 min at RT. Subsequently, monolayers were blocked in PBS + 1% BSA and 0.1% Triton X-100 (Sigma, SLCD3084) for 1 h at RT. Monolayers were incubated with commercially available primary antibodies to: DJ-1 (mouse-Abcam, ab11251, 1:250) and Adenovirus type 5 (rabbit-Abcam, ab6982, 1:500 overnight at 4°C in blocking buffer. Monolayers were incubated with secondary antibodies mouse-Alexa488 (1:1000) and rabbit-Alexa594 (1:1000) for 1 h at RT. Nuclei were stained using TO-PRO^®-3 iodide (1:10,000; Molecular Probes, Eugene, OR, USA) in PBS for 10 min. Transwells were mounted on microscope slides with Vectashield^® mounting medium (Vector Laboratories, Newark, CA, USA, H-1000-10). Monolayers were analyzed using a Leica laser scanning confocal microscope (model TCS-SP8, Wetzlar, Germany). Each individual xy image represents a three-dimensional projection of the entire monolayer (sum of all images in the stack).