Anti lc3 antibody

Primary neuronal cultures were treated with 50 µM NMDA (Tocris) for 5 min (Lee et al., 1998) or with 50 µM DHPG (Sigma–Aldrich) for 5 min. After 15 or 60 min, neurons were fixed with PFA (4%). Then, cells were washed three times for 5 min in 1x PBS. PFA was quenched with NH₄Cl 50 mM for 10 min. Cells were then permeabilized with 0.2% Triton X-100 for 5 min and incubated in the presence of BSA (1%) for 30 min. Cells were incubated by anti-LC3 antibody (Sigma) for 60 min at 37 °C. Unspecific staining was blocked by incubating coverslips in 1% BSA for 1 h at room temperature. Primary antibodies were revealed with Alexa 647 coupled secondary antibodies.

Recombinant human TGF-β, recombinant FGF-2 and recombinant human TNF-α were purchased from Biolegend (San Diego, CA). Rapamycin was from ENZO life science (Loerrach, Germany). Bafilolycin A1 was from Invivogen. FITC-PHA was from Vector (FL-1111). FITC-PNA was from Sigma (L7381). DAPI was from Sigma (D9542). Alexa Flour 488, 594 or 647 conjugated anti-mouse or anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The primary antibodies against JLP (ab12331, 1:1000), Fsp-1 (ab197896, 1:200), α-SMA (ab124964, 1:1000), Fibronectin (ab45688, 1:500), Collagen-I (ab34710, 1:1000), Ki67 (ab16667, 1:250), TGF-β (ab92486, 1:500), phospho-Smad2 (ab188334, 1:1000), phospho-Smad3 (ab52903, 1:1000) and p62 (ab56416, 1:200) were all from Abcam (Cambridge, MA). Anti-LC3 antibody (ab51520, 1:100, Abcam) was used for immuno-staining and Anti-LC3 antibody (L7543, 1:1000, Sigma) was used for western blotting, respectively. Anti-Nephrin and anti-F4/80 (123120, 1:100) antibodies were from Progen and Biolegend, respectively. Anti-Caspase-3 (#9664, 1:1000), anti-Beclin 1 (#3738,1:1000) was from Cell signaling Technology (Danvers, MA). Anti-GAPDH (sc-365062, 1:2500) was purchased from Santa Cruz (Santa Cruz, CA).

Cells were grown on glass coverslips (Fisher Scientific, Pittsburgh, PA, USA) and treated in the presence or absence of PP242 for 6 h. Coverslips were incubated with anti-LC3 antibody (Sigma), followed by AlexaFluor 488 labeled anti-rabbit antibody (Thermo Fisher Scientific, Rockford, IL, USA). Next, coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and analyzed via confocal microscopy (LSM-510, Carl Zeiss, Oberkochen, Germany).

Cells were scraped, lysed, and collected in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.002% bromophenol blue). Protein concentrations were determined using a protein assay kit (Bio-Rad DC; Bio-Rad, Segrate, Italy). Cell extracts were separated on 7.5% or 10% (w/v) homogeneous slab gels (40 μg of protein/lane) using SDS-PAGE and then transferred to nitrocellulose membranes (Protran; Whatman-GE Healthcare, Milan, Italy). Membranes were hybridized with a rabbit polyclonal anti-LC3 antibody (1:1,000 dilution, Sigma-Aldrich), mouse monoclonal anti-cytochrome C1 antibody (1:10 dilution, Santa Cruz Biotechnology), mouse monoclonal anti-beta tubulin or beta actin antibody (1:1,000 dilution, Santa Cruz Biotechnology) followed by an incubation with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1:10,000 dilution, GE Healthcare, Cologno Monzese, Italy). The relevant proteins were detected using chemiluminescence kits (Pierce EuroClone S.p.A., Pero, Italy), and the signals were acquired and analysed using an image acquisition system (Uvitec mod Alliance 9.7, Uvitec, Cambridge, UK). A mouse monoclonal anti-GAPDH antibody (1:5,000 dilution, Merck S.p.a., Vimodrone, Italy) was used as a loading control.

Anti-p62, anti-caspase-3, anti-Beclin-1, anti-Vps34, and anti-Atg5 antibodies were purchased from Cell signaling. Anti-LC3 antibody and necrostatin-1 were purchased from Sigma. Doxorubicin, chloroquine diphosphate (CQ), doxycycline, and cycloheximide were purchased from Calbiochem. zVAD was purchased from R&D system. 20(S)-ginsenoside Rg₃ was isolated by prep-LC. Glutathione S-transferase (GST)-TRAIL was obtained as described previously [67 (link)].

Autophagy in HeLa 229 cells was induced by nutrient-starvation or rapamycin treatment. For nutrient-starvation, HeLa 229 cells were washed and incubated with Krebs Ringer bicarbonate buffer pH 7.6 (KRB; 118.5 mM NaCl, 4.47 mM KCl, 1.18 mM KH₂PO₄, 23.4 mM NaHCO₃, 6 mM glucose, 2.5 mM CaCl₂, 1.18 mM MgSO₄, and 6 mg/l phenol red) for 0–6 h. For rapamycin treatment, cells were incubated with 1 µM rapamycin [stock 1 mM in dimethyl sulfoxide (DMSO), Sigma Aldrich, St. Louis, MO] in MEM for 4 h. Lysosomal protease inhibitors, 10 µg/ml E64d (Peptide Institute, Inc., Osaka, Japan) and 10 µg/ml pepstatin A (Peptide Institute, Inc.) were used to inhibit the lysosomal turnover. For immunostaining, the cells were fixed with 4% paraformaldehyde (Wako), washed with PBS, and lysed with 50 µg/ml digitonin (Wako). After quenching in 50 mM NH₄Cl, the cells were blocked in 2% (w/v) bovine serum albumin, 5% (v/v) normal goat serum in 20 mM Tris-HCl, 150 mM NaCl. Lysosomes were immunostained with anti-lysosomal-associated membrane protein 1 (LAMP1) antibody (Sigma Aldrich) and rhodamine-conjugated anti-rabbit immunoglobulin G (IgG) (MP Biomedicals, Irvine, CA), whereas autophagosomes were immunostained with anti-LC3 antibody (Sigma Aldrich) and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (MP Biomedicals). Fluorescence signal was observed under confocal microscope.