Amersham ecl prime solution

Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) in 10% to 14% SDS–PAGE gels. The polyacrylamide gels were then either imaged directly using a Typhoon 9200 Variable Mode gel scanner (GE Healthcare; Cy5 filter setting, excitation 633 nm, emission 670 nm, bandwidth 30 nm or GFP excitation 526 nm, emission 532 nm, short pass) or subsequently used for western blots. Proteins were blotted overnight at 4°C onto polyvinylidene difluoride (PVDF) membranes (Biorad) and subsequently, the membranes were blocked for 6 h at RT (or overnight at 4°C) with Tris‐buffered saline supplemented with skim milk powder and Tween‐20 (M‐TBST; 25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5% (w/v) skim milk powder, 0.05% (v/v) Tween‐20). Primary antibodies in M‐TBST were applied at 4°C overnight. After washing (1 × 5 min TBS, 2 × 5 min TBST, 3 × 5 min TBS), the blots were decorated for 1 h at RT with the respective HRP‐conjugated secondary antibody diluted in M‐TBST (at 10‐fold lower dilution than the respective primary antibody). Blots were imaged using Amersham ECL prime solution (GE Life Sciences) and a Fusion Pulse 6 imager (Vilber Lourmat). Quantifications were conducted using Bio‐1D software (Vilber Lourmat).

The whole protein extracts from the tissue samples were prepared using a PRO-PREP lysis buffer (#17081, iNtRon Biotechnology, Seongnam, Korea), and the protein concentrations were determined using a bicinchoninic acid protein assay (#23227, Thermo Fisher Scientific, Waltham, MA). The proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by blotting onto nitrocellulose membranes, and probing with the antibodies against HOXA11 (#SC-48542, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). The blotted membranes were then incubated with the goat anti-rabbit IgG secondary antibody for 1 hour. Subsequently, the membranes were incubated in an Amersham ECL-prime solution (#RPN2232, GE Healthcare Life Sciences, Pittsburgh, PA) in the dark for 1 minute and exposed under FluorChemHD2 (Cell Biosciences, Santa Clara, CA) for visualization. Only the samples with a consistent result from repeated experiments were selected for analysis. The densities of the bands were measured using free image analyzer software (ImageJ V1.8x, National Institutes of Health, http://rsb.info.nih.gov/ij/). The results are presented as the mean±standard error of mean calculated from independent samples.