Rabbit anti phospho ulk1

Rabbit mAb to LC3I antibody (12741, dilution 1/800), rabbit mAb to p62 (23214, dilution 1/1000), rabbit anti-Beclin-1 (3495, diluted 1/1000), rabbit anti-Phospho-ULK1 (14202, diluted 1/800) and ULK1 (8054, diluted 1/1000) were all purchased from Cell Signaling Technology, Inc. Rabbit polyclonal anti-LC3II antibody (ab63817, dilution 1/1000) was purchased from Abcam. Anti-actin was obtained from Epitomics, an Abcam company (Cambridge, MA, USA). miR-34a inhibitors (5’-AGCCUUGCUGCAGGUGCGCAU-3’) and a nonsense control (NC) sequence (5’-UGCCUUACUGACGGUCGGAGA-3’) were obtained from Shanghai GeneChem, Inc. (Shanghai, China). According to the protocol, Huh7-R and HepG2-R cells (1 × 10⁵) were transfected with 50 nM miR-34a inhibitors, 50 nM miR-34a mimics, 50 nM scramble control inhibitors or 50 nM scramble control mimics using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) for ~ 30 min at room temperature. After 48 h of transfection, the transfected cells were used for subsequent experiments. 3-Methyladenine (3-MA), a class III phosphoinositol 3-kinase (PI3K) inhibitor, and choroquine (CQ), were used as selective inhibitors of autophagy, was purchased from Santa Cruz Biotechnology (Texas, U.S.A). Rapamycin (rapa), a potent and specific mTOR inhibitor used as an autophagy agonist, was obtained from Selleck.

The following antibodies were used: rabbit anti-LC3B, mouse anti-MAP2, and FITC-conjugated goat anti-rabbit IgG (Sigma Chemical, St. Louis, MO); rabbit anti-beclin-1 and mouse anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-NeuN (Millipore, Temecula, CA); rabbit anti-phospho-AMPK (Thr172), rabbit anti-AMPK, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-mTOR, rabbit anti-phospho-ULK1 (Ser555), and rabbit anti-ULK1, rabbit anti-cleaved caspase 3, rabbit anti-caspase 3 (Cell Signaling, Danvers, MA); Texas red-conjugated goat anti-mouse IgG (Vector Laboratories, Burlingame, CA); Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories, West Grove, PA). 3-Methyladenine, rapamycin, bafilomycin A1, and monodansylcadaverine (MDC), wortmannin, LY294002 (Sigma Chemical) and Torin-1 (Tocris Bioscience, Ellisville, MO) were used in our autophagy flux studies. MDMA (purity, 98%) was obtained from the Investigation Bureau of Taiwan.