Western blot analysis after 2448 hours of transfection. The siRNA duplexes were synthesized by Bioneer and sequences for the siRNA used are shown in Preparation of cellular extract and Western blot analysis For the preparation of total protein, cytosolic and nuclear MedChemExpress Crenolanib fraction, RAW 264.7 macrophage cells were seeded in 35-mm dishes at a density 1 106 cells/well or 60 mm dish at a density of 3 106 cells/ well, respectively. After 24 hours incubation, cells were treated with gAcrp as indicated time points. Total proteins were then extracted using RIPA lysis buffer containing Halt protease and phosphatase inhibitor single-use cocktail. For cytosolic and nuclear fraction, cells were lysed using subcellular fractionation buffer. The cellular lysates were homogenized through 25 G syringes 1015 times followed by keeping on the ice for 20 minutes and then centrifuged at 3,000 rpm for 5 minutes at 4C to separate nuclear pellet. The supernatant was centrifuged at 8,000 rpm for 20 minutes and supernatant was taken as cytosolic fractions. For the preparation of nuclear fraction, nuclear pellet was lysed in RIPA buffer and protein was extracted as same process as total protein extraction. For immunoblot analysis, 2030 g of solubilized proteins were loaded and resolved by 815% SDS-PAGE. The proteins were then transferred to PVDF membranes, blocked with 5% skim milk in phosphate-buffered saline/ Tween 20 for 1 hour, incubated with the designated primary antibodies overnight at 4C, washed and incubated with the secondary horseradish peroxidase labeled anti-rabbit/ mouse antibody. Primary antibodies were diluted at a ratio of 1:2000 in 3% Bovine Serum Albumin. Chemiluminescent images of the blots were finally captured using a Fujifilm LAS-4000 mini. The PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19776696 membranes were then stripped and re-probed with desired antibodies as a loading control. Preparation of conditioned media Conditioned media was prepared by same methods as described previously. In brief, RAW 264.7 macrophages were stimulated with 1 g/ml of gAcrp for 8 hours, washed and then incubated for additional 12 or 24 hours in growth media without gAcrp. Supernatants were then collected as conditioned media. After centrifugation for 5 minutes at 5,000 g, the supernatants were mixed with fresh media at 2:1 ratio and used for incubation of RAW 264.7 macrophages. Media collected from the cells treated with media alone in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19778700 the absence of gAcrp was used as the control group. Confocal microscopic analysis For confocal microscopic analysis, RAW 264.7 macrophages were seeded at a density 5 104 cells/well in 8-well chamber slides. After overnight incubation, cells were transfected with enhanced green fluorescent protein -LC3 expression plasmid using Fugene HD transfection reagent as described previously. After 48 hour of transfection, cells were treated with gAcrp for the indicated time periods. Cells were then fixed with 4% paraformaldehyde solution and confocal images were taken using an A1 Confocal Laser Microscope System. Autophagic puncta was quantitated from confocal images from triplicate experiments. The images were 
expressed as percentage of cells with GFP-LC3 dots obtained from at least 100 cells with Image Inside software version 2.32. Statistical analysis Values were presented as mean S.E.M. from at least three separate experiments. Data were analyzed by one-way analysis of variance and Tukey’s multiple comparison tests using GraphPad Prism software version 5.01. Differences between groups were co
