Anti ctbp1

The antibodies used in this study are as follows: Anti-CtBP1 (1:5000, Abcam, Cambridge, MA, USA); β-Actin Rabbit mAb (High Dilution) (1:100,000, Abclonal Technology Co., Ltd., Wuhan, China); Goat Anti-Rabbit IgG H, and L (HRP) (1:500, Abcam, Cambridge, MA, USA); and Alexa Fluor™ Plus 488 conjugated secondary antibodies (Gibco, Invitrogen Co., Carlsbad, CA, USA).

The ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (CST) according to the manufacturer’s protocol. Immunoprecipitants were recovered overnight with specific antibodies or with normal rabbit IgG (CST). The purified DNA was amplified by PCR using the SP-B promoter-specific primers: Sftpb forward, 5′-CAGACAGAAGTCATCCTTGTTGAATG-3′; Sftpb reverse, 5′-ACATGGTACCGACTTGGCC-3′. PCR was performed under the following conditions: 95 °C for 5 min, followed by 30–35 cycles of 95 °C for 30 sec, 58 °C for 30 sec and 72 °C for 30 sec, and a final step of 72 °C for 5 min. The PCR products were subjected to 2% agarose gel electrophoresis. The following rabbit antibodies were used: anti-Histone H3 (CST; 1 μg/sample), anti-Pan-Methyl-Histone H3 (K9) (CST; 1 μg/sample), anti-Acetyl-Histone H3 (K9) (CST; 1 μg/sample), anti-CtBP1 (Abcam; 1 μg/sample), anti-Foxp1 (CST; 1 μg/sample), and anti-Foxp2 (Abcam; 1 μg/sample).

Total protein was extracted using the RIPA buffer with 1% PMSF (Beyotime Biotechnology). For the Western blot analysis, PVDF membranes were incubated with the following primary antibodies overnight at 4°C: anti-CTCF (Cell signaling Technology, Danvers, MA, USA), anti-Histone3 (Beyotime Biotechnology), anti-SRC (Abcam), anti-CTBP1 (Abcam), anti-SERPINE1 (Abcam) and anti-β-actin (Beyotime Biotechnology). After the samples were washed with 0.1% TBST, they were incubated with the appropriate secondary antibodies (Beyotime Biotechnology), and the Chemilmanger™ 5500 system (Alpha Innotech, San Jose, CA, USA) was used to visualize the results. The gray value was calculated using the Image J software (http://rsb.info.nih.gov/ij/).

Paraffin-embedded tissue sections were de-paraffinized with xylene, graded ethanol and ddH₂O. Antigen retrieval was achieved by incubating the specimens with boiled citrate buffer (10 mM, pH 6.0) for 15 minutes. The tissue sections were subsequently exposed to 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity. After blocking with goat serum for 20 minutes at room temperature, the sections were incubated at 4°C overnight with the following primary antibodies: anti-CTCF (Abcam, Cambridge, UK), anti-Ki-67 (Bioss Antibodies Inc., Woburn, MA, USA), anti-SRC (Abcam), anti-CTBP1 (Abcam), and SERPINE1 (Abcam). Then, the sections were incubated with HRP-labeled secondary antibodies at 37°C for 20 minutes and visualized with the DAB detection kit following the manufacturer’s instructions. The negative control samples were incubated with PBS in the absence of primary antibodies. The images were recorded with MCID, and the expression of positive cells was scored as previously described [30 (link)]. Briefly, each sample was assigned an intensity score (0-3) and a percentage of positive cells score (0=0%, 1=1-19%, 2=20-79%, 3=80-100%). An overall IHC score was calculated by multiplying the intensity score and the percentage value of positive cells.