Anti lc3b 2775

After treatment, cells were washed with PBS and lysed with RIPA buffer with protease inhibitors (Merck, New York) and phosphatase inhibitors (Merck). Equal amounts of protein (40 μg/lane) were resolved by SDS-PAGE, and transferred to PVDF membrane (Roche, Indianapolis, IN). All primary antibodies were incubated overnight at 4°C: anti-Bcl-2 (2870), anti-Bcl-xL (2764), anti-Mcl-1 (5453), anti-Bad (9239), anti-Bid (2002), anti-Bim (2933), anti-Puma (12450), anti-Bak (12105), anti-Bax (5023), anti-ATG5 (8540), anti-ATG7 (2631), anti-Beclin-1 (3738), anti-β-actin (4970), anti-cathepsin D (2284), anti-LC3B (2775), anti-p62 (8025), anti-ubiquitin (3936) (Cell signaling, Beverly, MA), anti-cathepsin B (C6243) (Sigma-Aldrich), and anti-cathepsin L (BMS1032) (eBioscience, San Diego, CA). All primary antibodies were used at a dilution of 1:1000, except for anti-ATG7 protein, which was used at a dilution of 1:500. The membranes were then incubated for 1 h with secondary peroxidase-conjugated antibodies (1:2000) (Anti-rabbit antibodies, Cell Signaling Technology, 7074; Anti-mouse antibodies, Cell Signaling Technology, 7076). Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology, 7003) and exposed to X-ray film (FUJIFILM Europe GmbH, Dusseldorf, Germany). The films were analyzed by densitometry with ImageJ software.

Anti-HO-1 (ab13243), anti-Nrf2 (ab89443), anti-NQO1 (A18; ab28947), anti-histone H3 (ab1791), anti-ubiquitin (ab7780), anti-SQSTM1/p62 (ab56416), and anti-Keap1 antibodies (ab139729) were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin antibody (sc-376421) and normal mouse immunoglobulin G (IgG) (sc-2025) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-tubulin (2128L) and anti-LC3B (2775) antibodies were purchased from Cell Signaling Technology (Beverly MA). Protein A + G agarose (P2012), mitochondrial membrane potential assay kits with tetrachloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) (C2006), and ROS assay kits (S0033) were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). Mitochondrial superoxide (MitoSOX) Red MitoSOX indicator for live-cell imaging (M36008) was purchased from Thermo Fisher Scientific (USA). Luteolin (T1027) was purchased from TargetMol (USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-rabbit IgG, HRP-linked antibody (7074S), anti-mouse IgG, and HRP-linked antibodies (7076S) were from Cell Signaling Technology (Beverly, MA).

Radioimmunoprecipitation assay buffer (RIPA) was applied to lyse cells. Total proteins were then harvested and quantified with bicinchoninic acid assays (Beyotime, Shanghai, China). The target proteins were separated through 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with nonfat milk, incubated with primary antibodies, and then incubated with secondary antibody diluted at a ratio of 1:10,000 (Jackson ImmunoResearch, West Grove, PA, USA). The primary antibodies were anti-LC3B (2775, Cell Signaling Technology, Beverly, MA, USA), anti-p62 (88588, Cell Signaling Technology), anti-HuR (ab28660, Abcam, Cambridge, MA, USA), anti-SFRS1 (ab133689), anti-FMRP (ab17722), anti-ALKBH5 (ab195377), anti-IGF2BP1 (ab82968), anti-LIN28A (ab46020), anti-FTH1 (ab65080), anti-NCOA4 (ab86707), anti-BCL-2 (ab32124), anti-BECN1 (ab62557), and anti-GAPDH (ab9485). The protein signals were visualized with enhanced chemiluminescence detection reagents (ECL, Millipore, Burlington, MA, USA) and quantified with Image Lab software (Bio-Rad, Hercules, CA, USA).