Rabbit anti cmyc antibody

TRI^® reagent was purchased from Sigma Aldrich; High-Capacity cDNA Reverse Transcription Kit, Power SYBR Green Supermix and TaqMan^® Gene Expression Master Mix from Applied Biosystems; antibodies were purchased from Santa Cruz Biotechnology, USA; ECL reagent (SuperSignal^® West Pico Chemiluminescent Substrate) was procured from Thermo Scientific, USA; rabbit anti-cMyc antibody was purchased from Cell Signaling Technology; tissue culture media, serum and additional reagents were procured from HIMEDIA; RNase and cloning kits were purchased from Fermentas; HiPerfect transfection and silencing reagents and QIAprep^® Spin Miniprep kit were procured from Qiagen. Restriction enzymes were purchased from Thermo Scientific, USA.

Chromatin immunoprecipitation (ChIP) assays were performed using SimpleChIP^® Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003) according to the manufacturer’s protocol. In brief, cells were first cross-linked by formaldehyde. Cells were then lysed, and chromatin was harvested and fragmented using enzymatic digestion and sonication to a length of approximately 150–900 base pairs. Fragmented chromatin was immunoprecipitated using rabbit anti-c-Myc antibody (Cell Signaling Technology, 13987) or rabbit immunoglobulin G (IgG) control and then extracted and purified. Two percent of precleared DNA (before addition of antibodies) was set aside as an input. Purified DNA was subjected to real-time PCR or standard PCR amplification using the primers, forward, 5′-GCCCCGCAGGTAGTCAGG-3′, and reverse, 5′-AGCCACGATTCTCTCCACG-3′.

MC3T3-E1 osteoblasts were plated at 3500 cells/cm² in 6-well culture dishes containing coverslips, previously cleaned and sterilised, and grown for 4 days to 80% confluence. At the end of each set of irradiations, the untreated and irradiated pre-osteoblasts were fixed in 4% paraformaldehyde (PFA) and permeabilised with 0.3% Triton X-100 as previously described [75 (link)]. The cultures were then incubated for 2 h at room temperature with the following primary antibodies: rabbit anti-c-myc antibody (1:100 dilution, Cell Signaling, Euroclone, Milano, Italy), rabbit anti-cyclin D3 antibody and rabbit anti-cyclin dependent kinase (CDK4) antibody (1:50 dilution, Santa Cruz Biotechnology, DBA, Milano, Italy). After washing, cells were incubated with Alexa Fluor-488 chicken anti-rabbit IgG or with Alexa Fluor 594 goat anti-rabbit IgG (1:100 dilution, Life Technologies, Monza, Italy) for 1 h at room temperature. The reaction controls were performed by complexing the primary antibody with a relative blocking peptide or by omitting the primary antibody. Coverslips were mounted on slides with phosphate-buffered saline (PBS)/glycerol (1:1). The slides were imaged using a fluorescent microscope and fluorescence analysis was performed using a Tecan Infinite fluorimeter with a 590 nm excitor filter and emission at 635 nm for Alexa Fluor 594 or 485, and of 535 nm for Alexa Fluor 488.