Ab56957

SAE1 expression levels between HGTs and PBTs were semi-quantitatively compared by immunohistochemistry (IHC) scoring [21 (link), 22 (link)]. Tissue sections were incubated with SAE1 antibody (ab56957, Abcam) at 4 °C overnight followed incubating with horseradish peroxidase-linked secondary antibodies at 37 °C for 40 min, then reacting with 3,3′-diaminobenzidine substrate solution (Dako Cytomation GmbH) and counterstaining with hematoxylin. Three independent fields at 20-fold magnification for positive cells were chosen to evaluate the immunostaining intensity and percentage. The immunoreactivity scores were measured the sum of immunostaining intensity multiplied by percentage of positive cells following our previous approaches [4 (link), 22 (link), 23 (link)]. The immunoreactivity score of SAE1 less than 4 was defined a low expression level, and the score more than 4 was defined a high expression of SAE1.

Proteins were separated on a 10% or 12% SDS-PAGE gel to test the target protein level using specific antibodies. The specific primary antibodies included SAE1 (ab56957 & ab185552, Abcam), SUMO1 (ab32058, Abcam), Akt (4961, Cell signaling), p-Akt (4060, Cell signaling), CDK2 (ET1602–6, HuaBio), Cyclin D1 (2922, Cell signaling), p21 (ab109199, Abcam), Bcl-2 (ET1603–11, HuaBio) and active Caspase-3 (ET1602–47, HuaBio), which were used to detect SAE1-invoved cell signaling pathway. The corresponding secondary antibody was subsequently incubated for 1 h to visualize signals at room temperature. The mouse anti-β-actin antibody (TA-09, ZSGB) was taken for signal normalization.

Colorectal tumour and matched normal tissues specimens from stage II and III colorectal cancer patients (n=51) were obtained from biopsy. After chemo and radiation therapy, tumour specimens were obtained from patients without complete pathologic response (non-pCR) (n=18). Archived specimens from patients with colorectal carcinoma (n=51), as well as archived normal colorectal tissue were subject to IHC staining using the following antibodies SAE2 (1:200, ab58451, Abcam), SAE1(1:100, ab56957, Abcam) and PIAS1(1:200, ab109388, Abcam). Omission of the primary antibody was set as a negative control. IHC staining was evaluated by two independent pathologists who were blinded to patients' clinical outcome. The QS was calculated for each slide based on intensity and percentage of staining area. The intensity of staining was scored semiquantitatively as negative (0), weak (1), intermediate (2) or strong (3). The percentage of staining area was scored as 0–4% (1), 5–19% (2), 20–39% (3), 40–59% (4), 60–79% (5) and 80–100% (6). Two independent pathologists calculated QS by multiplying the intensity score with percentage of staining area score and the average score was obtained for each slide. P values were derived using two-tailed Student's t-test and uncertainties were indicated as s.d. The study was approved by the Research Ethics Board at the City of Hope (IRB # 10132).