Anti p stat3 y705 antibody

Cellular lysates were prepared by suspending 1 × 10⁶ cells in 100 µl of RIPA lysis buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) that was supplemented with 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride. The cells were extracted on ice for 30 min. The proteins were electro-transferred to nitrocellulose membranes (Millipore Corp., Bedford, MA), and specific proteins were detected using chemiluminescence. Anti-PDK1 antibody (cat#: 10026-1-AP), anti-Akt antibody (cat#: 10176-2-AP), anti-p-Akt (S473) antibody (cat#: 66444-1-Ig), anti-SOD3 antibody (cat#: 14316-1-AP), anti- GAPDH antibody (cat#: 60004-1-Ig), anti-PRDX1 antibody (cat#: 15816-1-AP) were purchased from Proteintech Group, Inc, Rosemont, IL, USA. Anti-STAT3 antibody (cat#: 9139), anti-p-STAT3 (Y705) antibody (cat#: 9145), anti-cleaved caspase-3 antibody (cat#: 9664) were purchased from Cell Signaling Technology Inc., Danvers, MA, USA. All primary antibodies were diluted at 1:500.

The immunofluorescence method used in this study was described by Pandurangan et al. [23 (link)]. Paraffin-embedded colonic tissue sections with a thickness of 5 μm were deparaffinized in xylene and then rehydrated in a graded series of ethanol solutions. The slides were then blocked with 5% BSA in TBS for 90 min. The sections were then immunostained with rabbit anti-NF-κB (Santa Cruz Biotechnology, CA, USA) and anti-pSTAT3^Y705 antibody (Cell Signaling Technology, CA, USA) diluted 1 : 100 with 5% BSA in TBS and incubated overnight at 4°C. After the sections were washed three times with TBS, the slides were incubated with goat and rabbit DyLight 550 secondary antibody (Thermo Scientific, Rockford, IL, USA) diluted 1 : 200 with TBS and incubated in the dark for 120 min at room temperature. The sections were then washed with TBS and incubated with the nucleus-specific counterstain propidium iodide (Nacalai Tesque, Japan) or 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) to stain the cell nuclei. The slides were mounted in an Ultracruz hard-set mounting medium (Santa Cruz Biotechnology Inc., Dallas, TX, USA), coverslipped, and visualized under a FSX100 fluorescent microscope (Olympus, Tokyo, Japan).

MCF-7 cells were grown in 10-cm dishes to 60 to 70% confluence, deprived in 1% charcoal stripped FBS (CT) and then treated with vehicle or 10 nM IGF-1 for 24 h. Thereafter, cells were cross-linked with 1% formaldehyde and sonicated. Supernatants were immuno-cleared with salmon DNA/protein A-agarose (Millipore) and immunoprecipitated with anti-pSTAT3 (Y705) antibody or nonspecific IgG (Cell Signaling Technology). Pellets were washed, eluted with a buffer consisting of 1% SDS and 0.1 mol/L NaHCO3, and digested with proteinase K (Sigma Aldrich). DNA was obtained by phenol/chloroform extractions and precipitated with ethanol. The yield of target region DNA in each sample after ChIP was analyzed by qRT-PCR. The primer pairs for the human S100A7 promoter containing the putative 100 bp STAT3 binding located on the human S100A7 promoter are as follows: 5’-GCTCTTTGTCCAAACACACACA-3’ (Fwd) and 5’-GGCACTTCTAGAAAACGCAAAG-3’ (Rv). Data were normalized to the input for the immunoprecipitation.