Mouse anti lc3b

Primary human fibroblasts (IMR90 cells) were grown on glass cover slips, treated as appropriate, and fixed with 4% paraformaldehyde. Unspecific epitopes were blocked with 3% BSA before permeabilization with 0.1% Triton X-100 in PBS. The fixed cells were then incubated overnight with primary antibodies diluted in PBS containing 1% BSA: rabbit anti-LC3A (1:200, from Cell Signaling, Danvers, MA, USA; order number 4599), mouse anti-LC3B (1:200, from NanoTools, Teningen, Germany; order number 0260-100), rabbit anti-LC3C (1:200, from Cell Signaling, Danvers, MA, USA; order number 14736), guinea pig anti-p62 (1:400, from Progen, Heidelberg, Germany; order number GP62-C), rabbit anti-LAMP2 (1:1000, from BioCat, Heidelberg, Germany; order number WA-ABV10697.100). The cells were subsequently incubated with Cy2-, Cy3- and Cy5-coupled secondary antibodies (1:500, from Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature. Cell nuclei were counterstained with 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI, from Sigma-Aldrich, St. Louis, MO, USA). The cells were then analyzed and photographed using a confocal laser-scanning microscope (TCS SP5 from Leica Microsystems, Wetzlar, Germany). All pictures were taken with a 63x objective and 2x zoom at the highest possible resolution (2048 × 2048 pixels).

Cells were washed three times with ice-cold PBS and gently scraped in lysis buffer: 50 mM Tris–HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.27 M sucrose, 1 mM DTT, and complete protease inhibitors (Roche). Cell lysates were snap-frozen until use. Samples were spun at 10,000g for 10 min at 4°C, and the supernatant containing the cell lysate was collected. Protein concentration was determined by Bradford assay using different concentrations of BSA as protein standards. SDS–PAGE and Western blot analyses were performed using standard protocols. Primary antibody incubation was always performed overnight at 4°C, whereas HRP-conjugated secondaries were incubated for 1 h at RT. Western blot images were acquired using a ChemiDoc gel imaging system (Bio-Rad). The following antibodies and dilutions were used in this study: rabbit anti-p62 (1:1,000; Abcam), mouse anti-NBR1 (1:1,000; Abnova 6B11), mouse anti-ubiquitin conjugates (1:1,000; Enzo FK2), mouse anti-LC3B (1:500; Nanotools), mouse anti-GAPDH (1:10,000; Sigma-Aldrich), mouse anti-tubulin III-beta (1:1,000; BioLegend TUJ1), mouse anti-puromycin (1:20,000; Merck 12D10), mouse anti-strep-tag (1:2,000; QIAGEN), mouse anti-RFP (1:1,000; ChromoTek), goat anti-rabbit HRP (1:10,000; Dianova), goat anti-mouse HRP (1:10,000; Dianova).

The animals were subjected to anaesthesia before fixation at stage 40 by 2 h of incubation in methanol 100% at room temperature (RT). Samples were rinsed three times with PTw 1× (1× PBS, 0.1% Tween, pH 7.3) and then incubated overnight in 15% sucrose/PTW1X at 4°C, and then again incubated overnight in 30% sucrose/PTW1X at 4°C. Cryosections of the larvae were processed for immunostaining as follows: rehydrated in 1× PBS for 30 min, washed in PBS‐0.1% Triton X‐100 and treated with antigen retrieval solution [proteinase K 20 mg/ml (Sigma‐Aldrich, Germany) dissolved in 10 mM Tris pH 8.0, 1 mM EDTA (TE)] for 15 min at 37°C. Cryosections were then permeabilized with 0.5% Triton X‐100 in 1× PBS for 20 min at RT, rinsed in PBS 0.1% Triton X‐100 and moved to blocking solution [2% BSA, 2% serum, 2% DMSO in PBS‐0.1% Triton X‐100] for 30 min at RT. Cryosections were incubated with rabbit anti‐collagen type II (Rockland, 1:400) and mouse anti‐LC3B (Nanotools, 1:100) antibodies overnight at 4°C, then washed with PBS‐0.1% Triton X‐100 and incubated with secondary antibodies, Alexa‐594 anti‐rabbit IgG (1:500), Alexa‐488 anti‐mouse IgG (1:500; Thermo Fisher) for 1 h at RT. Nuclei were stained with DAPI (1:500).