Peroxidase conjugated anti rabbit igg

The extracted MCF-7 and MDA-MB-231 cells were split using cell lysates (KeyGEN Company) with protease inhibitor cocktail (Biotool, Houston, TX, USA). Whole-cell proteins were electrophoresed under the conditions in 12% polyacrylamide gels. The separated proteins were transferred to a polyvinylidenedifluoride membrane. Nonspecific binding was blocked with 5% skimmed milk for 2 h at 37 °C. The membrane was then incubated with PTEN (1 : 1500 diluted; Proteintech), p-Akt 308, p-Akt 473 and Akt antibody (1 : 1000 diluted; Abgent, San Diego, CA, USA) at 4 °C overnight. A GAPDH antibody (1 : 2500 diluted; Bioworld, Minneapolis, MN, USA) was used as a control. Next, the membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1 : 5000 diluted; Thermo, Boston, MA, USA). Images were obtained from multifunctional gel imaging system (ImageQuant LAS 500, General Electric, Fairfield, CT, USA).

RAW264.7 cells (1 × 10⁶ cells/well) were seeded into a 6-well tissue culture plate and cultured overnight. Pre-treatment and infection of cells were the same as in invasion assay. At 30 min pi, cell lysates were collected, protein concentrations were determined using Bradford protein assay and were boiled for 5 min in 2x Laemmli sample buffer (Bio-Rad Lab. Inc., USA). Immunoblot assay was performed with slight modifications as we previously described [11 (link)]. Briefly, membranes were incubated with phospo-specific antibodies against ERK, JNK and p38α (1:250, Cell Signaling Technology, Inc., USA) in 5% bovine serum albumin (GenDEPOT, USA) at 4°C overnight. Incubation with secondary antibody was done using peroxidase-conjugated anti-rabbit IgG (Thermo Scientific, USA; 1:1,000 dilution) at room temperature for 1 h. β-actin antibody was used as a loading control. Membranes were exposed to a Molecular Imager ChemiDoc XRS+ system machine (Bio-Rad Lab.). NHI ImageJ software (USA) was used to quantify the immunoblots.

Protein was isolated from HT29 cells, colons of mice and human intestinal resections, as previously described [35 (link)]. Western Blot was performed to analyze protein expression. SDS-PAGE gels were used and equal amounts of protein were loaded. Then, proteins were transferred to nitrocellulose membranes, which were further incubated with specific primary antibodies (detailed in Table 2) as well as the secondary antibodies peroxidase-conjugated anti-rabbit IgG (Thermo Scientific, Waltham, MA, USA, 1:5000) or anti-mouse IgG (Invitrogen, Waltham, MA, USA, 1:2000). Protein bands were detected with Immobilon^® Forte Western HRP Substrate (Millipore, Burlington, MA, USA) or Immobilon^® Crescendo Western HRP Substrate in AMERSHAM ImageQuant 800 (GE lifescience, Cornellà de Llobregat, Spain). To normalize protein bands, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping and Multi Gauge V3.0 software (Fujifilm Life Sciences, Cambridge, MA, USA) was used to quantify the densitometry of the bands.