Hspb8

The procedure has been described elsewhere.²⁵ (link) Eyes were extracted quickly and fixed in 4% paraformaldehyde for 1 hour before the anterior segments were removed. After they were dehydrated at 4°C in 10%, 20%, and 30% sucrose for 2 hours, 2 hours, and 8 hours, respectively, the retinas were embedded in optimum cutting temperature compound (Sakura Finetek USA, Torrance, CA, USA) and sectioned (14 °m) using a freezing microtome (Leica, Wetzlar, Germany). The retinal slices were blocked at room temperature for 2 hours with 5% bovine serum albumin and 0.5% Triton X-100 in PBS, then immunostained with primary antibodies as follows: HspB8 (Cell Signaling Technology), anti-GFP antibody (ab10145, 1:1000; MilliporeSigma, Burlington, MA, USA), LC3B (Cell Signaling Technology), and p62 (Cell Signaling Technology). Cryosections were incubated with Alexa Fluor 488 (#711-545-152, 1:500; Jackson ImmunoResearch Labs) and Alexa Fluor 594 (Jackson ImmunoResearch Labs). Finally, 4′,6-diamidino-2-phenylindole (DAPI) was used to label the cell nucleus of the retina. The sections were observed and photographed using an Olympus FV1200 confocal laser scanning microscope.

Crude cardiac or NRCM extracts were prepared, followed by immunoblotting, as described.26 Antibodies used were as follows: LAMP2 (Lysosomal Associated Membrane Protein 2), mouse monoclonal (Developmental Studies Hybridoma Bank, ABL‐93); LAMP1 (Santa Cruz Biotechnology, sc‐19992); anti‐LC3 (encoding for MAP1LC3B (Microtubule Associated Protein 1 Light Chain 3 Beta) subunit; Novus Biologicals, NB100–2220); SQSTM1 (Sequestosome 1) (Abcam, ab5416); TFEB (Bethyl Labs, A303–673A); HA (H6908; Sigma), p70S6K (phosphorylated Ribosomal protein S6 kinase beta‐1) (2708; Cell Signaling); phosphorylated p70S6K (9234; Cell Signaling); 4‐EBP1 (Eukaryotic translation initiation factor 4E‐binding protein 1) (9644; Cell Signaling); phosphorylated 4EBP1 (2855; Cell Signaling); phosphorylated mTOR (2974; Cell Signaling); mTOR (2983; Cell Signaling); histone H3 (9715, Cell Signaling); GAPDH (ab22555; Abcam); Hspb8 (3059S, Cell signaling); and ACTA1/α‐sarcomeric actin (Abcam, ab7799) or actin (Sigma, A2066). Protein abundance was normalized to actin or GAPDH protein expression and reported as fold change versus control.