Lc3b antibody

Primary antibodies used are as follows: α-synuclein monoclonal antibodies Ab274 and Ab62 from our laboratory,^{51 (link)}α-synuclein, AMPK, p-AMPK, and p62 antibodies (Cell Signaling Technology, Danvers, MA, USA), p62 (BD Biosciences, San Jose, CA, USA), secretogranin II (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), LC3B antibody (Abcam, Cambridge, UK), and β-actin antibody (Sigma-Aldrich Corp., St. Louis, MO, USA). A monoclonal antibody for LAMP2 (H4B4) developed by Drs August and Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA.
Retinoic acid, poly-L-lysine, trehalose, sorbitol, sucrose, maltose, rapamycin, CQ, trehalase, invertase, and acarbose were also purchased from Sigma. BafA1 was purchased from EMD Millipore (Billerica, MA, USA). pLVX-mCherry-Gal3 plasmid was a kind gift from Dr. Edward M Campbell.^{52 (link)}

One million BON and NCI-295R cells per well were seeded in 6-well plates and treated for 2 and 6 h, respectively, with TNFα on the following day. Cells were lysed in the RIPA buffer containing Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The protein concentration was measured using the BCA kit (Thermo Fisher) with the PowerWave340 plate reader (Biotek, Winooski, VT, USA). With 10 µg of proteins loaded per well on the 3.9–20% gel, PAGE was performed for 2 h 30 min at constant voltage of 30 mA per gel in Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, Hercules, CA, USA).
The eBlot transfer system (Genscript, Piscataway Township, NJ, USA) was used for protein transfer on NC membranes. After blocking with NET-G buffer for 1 h, the membranes were incubated with LC3B antibody (Abcam) at +4 °C overnight, washed, and incubated with the HRP-labeled anti-rabbit antibody (GE Healthcare, Chicago, IL, USA) for 1 h at RT. For visualization, Lumi-Light ECL (Roche) and the LAS3000 imager (Fujifilm, Minato, Tokyo, Japan) were used. Subsequently, membranes were washed, blocked with NET-G, and incubated with mouse β-actin antibody (Sigma-Aldrich) at +4 °C overnight. As the secondary antibody, HRP-labeled anti-mouse antibody (GE Healthcare) was used. β-actin was visualized with Lumi-Light ECL in LAS3000.

HSA was obtained from CSL Behring AG (Switzerland). CQ was obtained from J&K Scientific Ltd. (China). Potassium permanganate (KMnO₄) and hydrogen peroxide (H₂O₂, 30 wt%) were obtained from Sino pharm Chemical Reagent Co. (China). 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Molecular Probes (USA). RPMI-1640 medium was obtained from Gibco (Grand Island, NY, USA). The LC-3B antibody, p62 antibody, and HIF-1α antibody were obtained from Abcam (UK). The pH-sensitive fluorescent dye SNARF-4F was obtained from Life technologies (USA). The hypoxia probe pimonidazole (Hypoxyprobe-1 plus kit) was obtained from Hypoxyprobe Inc. (USA), and 1×Phosphate buffer solution (PBS) and deionized water were used in the experiments. All male Babl/c nude mice (17~20 g) were obtained from Yangzhou University Medical Center.

Total protein was extracted using a combination of RIPA buffer, phenylmethylsulfonyl fluoride, and phosphatase inhibitors. The protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to poly(vinylidene fluoride) membranes. The membranes were blocked in 5% skim milk at room temperature for 1 h and then incubated with the designated primary antibodies at 4°C overnight. The excess primary antibody was washed off with Tris-buffered saline with Tween 20, and the corresponding secondary antibody was incubated at room temperature for 1 h. A United Kingdom UV imaging system was used for exposure imaging. ImageJ was used to analyze the experimental results. Western Blot antibodies:ATM antibody, Cell signaling (#2873); Phospho-ATM (Ser1981) antibody, Cell signaling (#13050); LC3B antibody, Abcam (AB192890); SAPK/JNK Antibody, Cell signaling (#9252); Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) antibody (#4668); AMBRA1 antibody, AB clonal (A12578); GAPDH antibody, AB clonal (AC002).

For mitochondrial staining, macrophages were incubated with MitoTracker® Red (Thermo Fisher, Waltham, MA, USA) at 37°C for 20 min. Then, cell slides or frozen tissue samples were fixed with 4% paraformaldehyde for 15 min at 4°C. After washing with PBS three times, cells were punched with 1% Triton and blocked with 2% horse serum for 1 hour at room temperature. Then, slides were incubated with primary antibody (LC3B antibody, 1 μg/ml; NLRP3 antibody, 1/200 dilution; and CD68 antibody, 1/100 dilution, all from Abcam) at 4°C overnight. After being washed with PBS three times, samples were incubated with the corresponding IgG-FITC or Rhodamine-conjugated secondary antibody (dilution factor 1 : 200) for 2 h at room temperature. Cell nuclei were counterstained by DAPI (1 mg/ml) for 5 min. After a final wash, the slides were visualized under a laser scanning confocal microscope (Olympus FV1200, Olympus, Tokyo, Japan).