Anti lc3 nb100 2220

For total protein extracts, cells were lysed with cold lysis buffer for 30 min on ice [10 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM DTT, 1 mM NaF, 0.5% NP-40, 10 mM sodium orthovanadate/complete protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada)]. The cytoplasmic/nuclear extracts were prepared as previously described by our group [24 (link)]. Protein extracts were analyzed by Western blot using SDS-polyacrylamide gels (10 or 12.5%) and transferred onto nitrocellulose membranes (Biorad), and signal was revealed using ECL (GE Healthcare, Piscataway, NJ, USA). Membranes were probed with anti-β-Actin antibodies as a loading control. GAPDH was used as a purity indicator for nuclear extracts. The antibodies used for protein expression analyses were: anti-RelA (sc-8008), anti-RelB (sc-226), anti-PARP-1/2 (sc-7150) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-p100/p52 (05–361) (Upstate Biotechnology, Charlottesville, VA); anti-LC3 (NB100-2220) (Novus biological, Oakville, ON); anti-GAPDH (AB9485-100) and anti-β-Actin (AB6276-100) (Abcam, San Francisco, CA).

Whole cell lysates were prepared with protein sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% Bromophenol blue) (BioRad, Hercules, CA). After separation in 10–15% SDS-PAGE, proteins were transferred onto polyvinylidene fluoride membrane and (Bio-Rad, Hercules, CA). The membranes were blocked with 4% skim milk in TBST for 1hr and then incubated with specific primary antibodies overnight at 4°C. Anti-ATG5 (ab54033, 1:2000) antibody was purchased from Abcam (Cambridge, UK); anti-LC3 (NB100-2220, 1:10,000) antibody was purchased from NOVUS Biologicals (Littleton, CO); p62 (Sc-28359) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Actin (MAB1501, 1:10,000) antibody was purchased from Millipore (Temecula, CA). For protein detection, the membranes were incubated with HRP-conjugated secondary antibodies and signals were detected with Super-signal West Dura HRP detection kit (Pierce, Rockford, IL).

A 30 µg denatured protein sample was loaded onto each well of a separating gel for SDS-PAGE electrophoresis (10% SDS-PAGE gels). Electro-transfer of proteins from gel to an Immobilon-FL PVDF membrane (Millipore) took 2 h in prechilled buffer with cold pack using 250 mA. Membrane blocking involved incubation with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween 20 (Bio-Rad Laboratories) for 1 h at room temperature. Primary antibody incubation with anti-LC3 (NB100-2220, Novus Biologicals, Littleton, CO, dilution 1:1000), anti-LAMP2 (sc-18822, Santa Cruz Biotechnology, Dallas, TX, USA, dilution 1:2000), anti-cathepsin D (sc-377299, Santa Cruz Biotechnology, dilution 1:500), anti-TFEB (D2O7D, Cell Signaling Technology, Davers, MA, USA, 1:500), and β-actin (AC-15, Thermo Fisher Scientific, dilution 1:2000) was performed overnight at 4 °C. The washed membrane was incubated with horseradish peroxidase HRP-conjugated secondary antibodies including anti-mouse IgG (H + L, A16066) and anti-rabbit IgG (H + L, A16110; Thermo Fisher Scientific, dilution 1:2000) for 1 h, washed, and developed by incubation with ECL substrate solutions for 5 min. The western blot images were acquired using the Chemidoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

All lysates were prepared with 2× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Total protein was measured using the Bradford assay (Bio-Rad) according to the manufacturer’s instruction. Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After blocking with 4% skim milk in tris-buffered saline supplemented with Tween-20, the membrane was incubated with primary antibodies including anti-MITF (MS-771-P1; Neomarkers), anti-TYR (a gift from Amorepacific Research Group), anti-LC3 (NB100-2220), anti-ATG5 (NB110-53818) and anti-actin (NB600-501; NOVUS Biologicals, Littleton, CO, USA), and anti-GFP (sc-9996; Santa Cruz Biotechnology, Dallas, TX, USA). For protein detection, the membranes were incubated with HRP-conjugated secondary antibodies (Pierce, Rockford, IL USA).

The following antibodies were used for immunoblotting, immunoprecipitation, and immunofluorescence staining: Anti-p62 (sc-25575), anti-Notch1 (sc-6014), anti-Fbw7 (sc-21185), anti-Beclin1 (sc-11427), anti-RBP-Jk (sc-28713), anti-β-actin (sc-47778), anti-GFP (sc-8334), anti-MEKK1 (sc-252), normal rabbit IgG (sc-2027), and normal goat IgG (sc-2028) were purchased from Santa Cruz biotechnology. Anti-LC3 (NB100-2220) was purchased from Novus Biologicals. Anti-cleaved Notch1 (Val1744) was purchased from Cell Signaling. Anti-Flag (A2220) was purchased from Sigma. Anti-phospho-ser/thr-pro was purchased from Upstate Biotechnology. Anti-Myc (9E10) and anti-HA (12CA5) were purified by agarose resin. Rapamycin and MG132 were obtained from Cayman Chemical and 3-MA from Acros Organics. BFA1 was obtained from Wako Reagent. DAPT and Chloroquine were purchased from Sigma.

Cells were trypsinized and lysed by sonication with RIPA buffer containing a protease inhibitor cocktail (Complete, 11697498001, Roche, MA, DE). Protein was quantified by Bradford assay. A total of 30 μg of protein were resolved by a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (PVDF) (IPVH00010, Merck Millipore, Cork, IRL). Membranes were blocked with 5% milk in PBS-Tween 20 (Sigma, p1379) for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies: anti-ATG7 (8558, 1:500, Cell signaling, MA, USA), anti-beclin-1 (3738, 1:1000, Cell signaling), anti-LC3 (NB100-2220, 1:1000, Novus Biologicals, CO, USA), anti-actin (A5441, 1:10,000, Sigma) or anti-VDP/P115 (PA5-30281, 1:1,000, ThermoFisher, IL, USA). Secondary antibodies used were HRP-linked anti-rabbit IgG (7074, 1:20,000, Cell signaling) and HRP-linked anti-mouse IgG (A2304, 1:20,000, Sigma). Immobilon™ Western (WBKLS0500, Millipore) detection reagents and a C-Digit Blot Scanner (LI-COR Biosciences, Lincoln, NE, USA) imaging system were used to visualize and digitalize the Western Blot images. Densitometric analysis was performed using Image Studio Lite Ver 5.3 software. The molecular weight was evaluated with pre-stained protein ladder, 10–180 kDa (26616, ThermoFisher, IL, USA).