Rabbit polyclonal anti lc3

1 × 10⁵ cells were placed on a poly-L-Lysine slide (Thermo Fisher Scientific, USA) and incubated at 37 °C to allow cell attachment to the slide. Cells were rinsed with 1X PBS and then fixed using a solution of 4% PFA/1X PBS [27 (link)]. Fixed cells were washed with 1X PBS and treated with a blocking solution (5% normal goat serum in 0.1% Tween-PBS) for 1 h to block unspecific protein binding sites; cells were then incubated o/n at 4 °C with primary antibodies: rabbit polyclonal anti-LC3 (1:250 dilution; Sigma-Aldrich), mouse monoclonal anti-PINK1 (1:250 dilution; Abcam, UK). Cells were washed with 1X PBS and incubated at RT for 1 h with secondary antibodies: CFTM 594 goat anti-mouse (1:700 dilution, Sigma-Aldrich, Italy) and CFTM 488A goat anti-rabbit (1:700 dilution; Sigma-Aldrich, Italy). Both primary and secondary antibodies were prepared in the blocking buffer. Finally, samples were washed with 1X PBS, mounted with Prolong^® Gold antifade reagent with DAPI (Thermo Fisher Scientific, USA), dried, nail-polished, and analyzed by confocal microscopy (Leica Microsystems Srl, Wetzlar, Germany).

The hATG5 and hATG16L1 sequences were cloned into the peGFP-C1 plasmid (Clontech), thus forming pGFP-ATG5 and pGFP-ATG16L1, respectively. The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12DN) constructs were kindly provided by Dr. Adi Kimchi39 (link). The PTM vector for the expression of HCV nonstructural proteins NS3 to 5B (pTM-NS3-5B) was kindly provided by Dr. Volker Lohmann. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5, mouse monoclonal anti-Flag, and mouse monoclonal anti-β-actin antibodies were purchased from Sigma Aldrich. Rabbit polyclonal anti-ATG12 and anti-ATG7 were purchased from Cell Signaling. Rabbit polyclonal anti-ATG16L1 antibody was purchased from MBL. Mouse monoclonal anti-HA was purchased from Roche. Mouse monoclonal anti-NS3 and anti-NS5A antibodies were purchased from BioFront. Rabbit polyclonal anti-NS3 and NS5A were obtained from Dr. Olivier Nicolas. Rabbit polyclonal anti-NS4B and anti-NS5B antibodies were kindly provided by Drs. Kouacou Konan and Takaji Wakita, respectively. Mouse monoclonal anti-GAPDH was purchased from Santa Cruz.

CHO-K1 cells were treated as indicated and lysed with RIPA Buffer (150 mM NaCl, 1% Triton x-100, 50 mM Tris pH 7.5, 1% Sodium Deoxycholate, 0.1% SDS, 2 mM EDTA, 50 mM NaF). Bradford assays were performed to determine the protein concentration in the obtained samples. Samples were run in polyacrylamide gels and transferred to PVDF membranes (BioRad). Membranes were blocked during 30 minutes with a blocking solution (10% BSA, 0.05% Tween 20 in PBS) and washed twice with 0.05% Tween 20 in PBS. Afterward, they were incubated with specific antibodies diluted in 0.05% Tween 20 in PBS Tween: 1 μg/ml rabbit polyclonal anti-LC3 (Sigma-Aldrich), or 0.1 μg/ml rabbit monoclonal anti-actin (Developmental Studies Hybridoma Bank, University of Iowa, USA) overnight at 4°C. Membranes were incubated with secondary antibodies conjugated with peroxidase (Sigma-Aldrich). Detection of immunoreactive bands was performed by chemiluminescence in a FluorChem Q imaging system (Protein Simple).