Normal rabbit igg

Mouse anti-Flag (F1804) and rabbit anti-HA (H6908) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). Rabbit anti-GFP (50430-2-AP), Rabbit anti-LC3 (14600-1-AP) and Rabbit anti-P62 Polyclonal antibody(18420-1-AP) were purchased from Proteintech Group (Chicago, IL, USA). Rabbit anti-β-actin (AC026) and anti-EV71-3D polyclonal antibodies were produced by ABclonal Technology (Wuhan, China). Rabbit anti-EV71-VP1 (PAB7631-D01P) polyclonal antibody was purchased from Abnova (Taipei city, Taiwan). Rabbit anti-Beclin1 (381896) antibody was purchased from ZENBIO (Chengdu, China). Delbecco modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Lipofectamine 2000, normal rabbit IgG and normal mouse IgG were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Protein ladder (26616) was purchased from Thermo scientific (Rockford, IL, USA). Complete, EDTA-free Protease Inhibitor Cocktail was purchased from Roche (Basel, Switzerland). Chloroquine diphosphate were purchased from Selleck (Houston, TX, USA). Rapamycin (53123-88-9) was obtained from Target Mol (Shanghai, China).

Chromatin immunoprecipitation (ChIP) was carried out using ChIP Assay Kit (Millipore, MA, USA) with the manufacturer's instructions slightly modified. The solutions used were from the ChIP Assay Kit unless otherwise specified. Anti-DNMT1 antibodies were subsequently added and incubated overnight at 4°C in a shaking incubator. Normal rabbit IgG acquired from Invitrogen served as negative control. Precipitates were evaluated by PCR for Fibulin-5 with the following primers 5′-GCTAAGCAAAACCAGGTGCT-3′ and 5′-GTGCGAAGGCGAGAAGAAA-3′ [21 (link)].

C2C12 myoblasts were cultured in growth or differentiation conditions and treated with RA or vehicle for 24 h as indicated. Chromatin immunoprecipitation (ChIP) analysis was performed as described [30 (link)] using antibodies for C/EBPβ (C-19; Santa Cruz Biotechnology), RAR (M-454; Santa Cruz Biotechnology), or non-immunized normal rabbit IgG (Invitrogen) as control, incubating at 4°C overnight. After sonication, DNA fragments were purified using the QIAquick PCR Purification Kit (Qiagen) and amplified by qPCR. Primer sequences for the Pax7 promoter were as follows: forward 5′-CCCGAACTGGCCCCCTTTCC-3′ and reverse 5′-TCCCCCGGAGGACTGGAACG-3′. Primer sequences for the intronic RARE region of the Smad3 promoter were as follows: forward 5′-ATGACTTGTTCCTGTCCTTC-3′ and reverse 5′-GCTAGGCAGAGTTCCCAGAA-3′. Primer sequences for the Smad2 promoter were as follows: forward 5′-AAGTCCCTGGAGGGAATGGA-3′ and reverse 5′-CACTGTAGGCAGAGCAGGTT-3′.

ChIP assays was performed according to the manufacturer’s protocol (56383 SimpleChIP^® Plus Sonication ChIP Kit 4 and RT Reagents, Cell Signaling Technology). Briefly, 100–150 mg Chinese cabbage leaves were cut into small pieces and divided and cross-linked with 1% formaldehyde at room temperature for 30 min. The crosslinking was terminated by adding 100 μl of 10 × glycine, and then the sample was mixed and incubated on ice for 5 min, washed with the mixture of PBS and 200 × protease inhibitor cocktail (PIC, twice every 10 min), and centrifuged. The supernatant was removed, and the tissue was resuspended in 1 ml mixture of 1 × ChIP sonication cell lysis buffer and PIC. After lysate was sonicated and centrifuged, 50 μl supernatant was taken for chromatin digestion. 10 μl digested chromatin was used as the “Input”, and the rest was subjected to the following immunoprecipitation experiments. Normal rabbit IgG (Invitrogen) was used as the negative control. ChIP PCRs were performed using primers that flank the G-box-like motif sites and primers that do not flank the sites in the promoter regions of target genes as controls. Primers (Comate Bioscience Technology) for ChIP are listed in Supplementary Table S2. The quantitative analysis of the ChIP gel electrophoresis map was carried out by Image J (National Institutes of Health).