Odyssey fc software

Eight-week-old female BALB/c
nude mice obtained from Charles River Laboratories (Germany) were
acclimated for at least for 24 h before tumor cell injection. SMYD2-engineered
KYSE-150 cells (4 × 10⁶) were resuspended in 100%
Matrigel (100 μL) and injected subcutaneously into the right
flank region of the mice. Treatment was started when tumors reached
a tumor area of 60–70 mm² (day 7 after inoculation).
Mice (n = 12 per group) were treated with 10, 30,
70, or 100 mg/kg (S)-4 po qd for 3 days,
or vehicle (PEG 400/water 8:2) po qd for 3 days. After the treatment
period, tumor samples were immediately frozen in liquid nitrogen and
stored at −80 °C. Frozen tumors were mechanically homogenized
using TissueLyser and stainless steel beads (Qiagen), and proteins
were extracted as described for the Western blot method. Whole protein
lysate (50 μg per sample) was transferred with the Dot-Blot
system MiniFold-1 (Whatman) onto a nitrocellulose membrane (Invitrogen).
Membrane were blocked in 5% milk PBS-T and immunoprobed with the SY46
methylation antibody. The secondary antibody used was goat antirabbit
IRDye 800 CW (LI-COR, no. 926–32211, 1:1000). Bands were detected
and quantified with Odyssey Fc software (LI-COR Biosciences).

Cells were lysed in
RIPA buffer Pierce (Thermo Fisher Scientific) with benzonase (Merck)
and protease inhibitors (Roche Diagnostics). Proteins were separated
by SDS-PAGE and blotted onto nitrocellulose membranes (Invitrogen).
Membranes were blocked in 5% milk PBS-T (phosphate buffered saline
with 0.1% Tween 20) and immunoprobed with antibodies raised against
histone H3 (Cell Signaling Technology no. 4499S), p53 (BD Biosciences
no. 554294), AHNAK (Santa Cruz Biotechnology, no. sc-390743, 1:500),
SMYD2 (abcam, no. ab108217, 1:1000), beta-actin (abcam, no. ab8224,
1:1000), or with the methylation specific antibody SY46 (Eurogentec).
The secondary antibodies used were goat antimouse/rabbit IRDye 800
CW (LI-COR Biosciences) and Alexa Fluor 680 goat antimouse/rabbit
IgG. Bands were detected and quantified with Odyssey Fc Software (LI-COR
Biosciences). Western blot assay for p53 methylation in HEK293T cells
was performed as described elsewhere with an antibody directed against
p53K370me1 kindly provided by Dr. Shelley Berger.^{13 (link),28 (link)}