Ab25631

Tissues were lysed using 300 μl RIPA plus 3 μl protease inhibitor PMSF (Beyotime, Beijing, China) on the ice. After centrifugation for 5 min at 12,000 × g, the supernatant was stored at −20°C. The protein concentration was determined using a BCA protein assay kit (Beyotime). Total proteins in the supernatant were subjected to separation in 10% SDS-PAGE, followed by transference onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk powder lasting 1 h at room temperature. Afterwards, the membrane was incubated by primary antibodies at 4°C overnight, and incubated with corresponding HRP-conjugated secondary antibodies (1:2,000; Abcam) for 50 min at room temperature in the dark. The primary antibodies included anti-Beclin1 (1:2,000; ab622557; Abcam), anti-LC3 (1:1,000; ab51520; Abcam), anti-LAMP2 (1:500; ab25631; Abcam) and anti-GAPDH (1:8,000; ab9485; Abcam). GAPDH was used as an internal control. The protein blot was visualized using Odyssey CLx.

Western blots were performed and analyzed as previously described [22 (link)]. Briefly, gastrocnemius muscle samples were homogenized in RIPA buffer, and then centrifuged at 12000 × g for 30 min at 4 °C. The quantification of protein in supernatant was accomplished using a BCA kit (Beyotime Biotechnology, Wuhan, China). The protein samples were boiled in the presence of sample buffer at 95 °C for 3 min. The proteins were subjected to the separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred onto a nitrocellulose membrane. The target protein was probed by a corresponding antibody and then visualized by enhanced chemiluminescence (ECL) reagent and imaged by the chemiluminescence imaging system Amersham Imager 680 (General Electric Company, USA). Antibodies used for Western blots were: Anti-Pax7 (Abcam, USA, ab199010), Anti-alpha smooth muscle Actin (Abcam, USA, ab5694), Anti-LAMP2-Lysosome Marker (Abcam, USA, ab25631), Anti-Beta actin (Abcam, USA, ab8226), Anti-Cleaved LC3B (Sigma, Germany, L7S43), Anti-ARPC5/p16 ARC (Abcam, USA, ab51243), p53 (1C12) Mouse mAb (Cell Signaling Technology, USA, 1C12). Origin images of all western blot have been uploaded as a single ‘Supplemental Material’ file.

Cells were cultured on sterile crawl plates and subjected to the indicated treatments. Immunofluorescence assays were performed after the indicated treatment time. The medium was aspirated from the wells and washed 3 times with PBS solution for 3 min each time; 4% paraformaldehyde was used to fix the cells at room temperature for 15 min. Then, 0.5% Triton X-100 (PBS preparation) was used to permeabilize the cells at room temperature for 20 min, and immunofluorescence blocking solution was used to block the cells at room temperature for 30 minutes, followed by incubation with anti-TFEB (1:100, 4240, CST), PP1 (1:200, 67070-1-Ig, Proteintech), or PPP1R9B (1:100, 55129-1-AP, Proteintech), LAMP2 (1:100, ab25631, Abcam), TFE3 (1:100, 14480-1-AP, Proteintech), MITF (1:100, 13092-1-AP, Proteintech), Cathepsin B (1:200, 12216-1-AP, Proteintech) overnight at 4 °C. After incubation, the cells were washed three times with PBST solution for 3 min each time, and the fluorescent secondary antibody was added and incubated for 1 hour before nuclei were restained with DAPI (Invitrogen, USA). Fluorescent images were observed under a fluorescence microscope and photographed (Olympus, BX53).

Using RT‐PCR we evaluated the expression of autophagy‐related genes including LAMP2, Beclin, LC3, Parkin. Additionally LAMP2 amount in investigated cells was evaluated using flow cytometer. Briefly, cells were detached from culture plate and centrifuged (350 × g for 5 min.) followed by the 10 min. fixation in 4% ice cold PFA. After washing, cells were then incubated in 0.1% Tween‐20 in HBSS for 20 min. After another wash, cells were incubated with anti LAMP2 antibody (ab25631; Abcam) diluted 1:200 in HBSS containing 10% Goat Serum for 30 min. at 22°C. Next cells were washed again and incubated with Alexa 488 goat anti‐mouse secondary antibodies (1:500, Alexa Fluor 488, ab150113; Abcam) for 30 min. at 22°C. At least five thousand stained cells were acquired and analysed by FACS Calibur flow cytometer. The samples were analysed using CellQuest Pro software. Moreover, using the same anti‐LAMP2 antibody, immunofluorescence staining of investigated cells was performed. Additionally, mitochondria were counterstained with MitoRed to investigate mitophagy. Cell's nuclei were stained with DAPI. Cells were observed and photographed using confocal microscopy (Observer Z1 Confocal Spinning Disc V.2 Zeiss with live imaging chamber). The fluorescence intensity was calculated using ImageJ software. Using TEM imaging we also visualized autophagosomes formation (FE‐STEM Auriga60).

For immunofluorescence colocalization between lysosomes and mitochondria, Hep3B cells were seeded on gelatin-coated coverslips. After treatments, cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 15 min, washed with PBS and permeabilized with 0.2% saponin (Sigma) in PBS and 1% fatty acid-free BSA (Sigma), all at room temperature. After washing cells with PBS, they were stained overnight at 4 °C with the [H4B4] LAMP2 (ab25631, Abcam) and the Tom20 (sc-11415, Santa Cruz Biotechnology) antibodies. Afterward, cells were washed with PBS and incubated for 1 h at room temperature with Alexa 488-conjugated antimouse (Z25002, Molecular Probes, Eugene, OR, USA) and Alexa 647-conjugated antirabbit (Z25308, Molecular Probes) IgGs. Coverslips were washed with PBS and mounted on glass slides with fluorescent mounting medium Fluoroshield™ containing 4′6-diamidino-2-phenylindole (DAPI) (Sigma) to be visualized in a Zeiss LSM 800 confocal laser scanning microscope (Zeiss AG, Jena, Germany) and samples were analyzed using ZEN software (Zeiss AG, Jena, Germany).