Ab99273

Immunohistochemistry (IHC) was executed to determine IPO7 protein expression in 55 pairs of pancreatic cancer tissues and adjacent tissues. The specimens were fixed in 10% formaldehyde and embedded in paraffin. Next, the paraffin block was sliced, and the sections were dewaxed and rehydrated. Antigen retrieval was performed in a microwave by placing the sections in epitope retrieval solution (0.01 M citrate buffer, pH 6.0) for 5 min; endogenous peroxidase was inhibited by immersing the sections in 0.3% hydrogen peroxide for 10 min. Then, the sections were blocked in 5% bovine serum for 30 min. Then, anti-IPO7 antibody (ab99273, Abcam, 1:100) was supplemented to incubate the sections at 4°C for 12 h in a humidified box. Subsequently, the sections were rinsed with phosphate buffered saline (PBS) and then incubated with a biotin-linked antiserum for 1 h. Next, the sections were rinsed again and stained with 3,3-diaminobenzidine. Finally, the sections were observed under a microscope, and the staining was examined by two independent pathologists.

Extraction of total protein was performed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Then, the protein was quantified using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States). Following that, the proteins were mixed with loading buffer, heated in boiling water for 10 min for denaturation, dissolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, United States). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies [anti-IPO7 antibody, ab99273, Abcam, 1:500; anti-E-cadherin antibody, ab231303, Abcam, 1:500; anti-N-cadherin antibody, ab76011, Abcam, 1:500; anti-N-cadherin antibody, ab92547, Abcam, 1:500; anti-Snail antibody, ab216347, Abcam, 1:500; anti-alpha-smooth muscle actin (aSMA) antibody, ab184705 Abcam, 1:500; anti-GAPDH antibody, ab8245, Abcam, 1:2,000] overnight at 4°C. The membrane was rinsed and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) for 2 h at room temperature. Ultimately, the protein bands were developed with an enhanced chemiluminescence (ECL) kit (Biossci, Wuhan, China).

10.10⁶ cells were harvested, washed with PBS, and resuspended in lysis buffer (400 mM NaCl, 10 mM HEPES, pH 7.5, 0.1% NP‐40, Sigma P8340); the NaCl concentration was then brought down to 150 mM; and samples were sonicated using a probe sonicator and then centrifuged at 17,000 rcf in order to remove insoluble material. The protein concentration of the lysates was measured using a BCA assay (Thermo Fisher, 23225), and equivalent protein quantities were then incubated with 50 μl of pre‐washed anti‐HA agarose beads (Sigma, A2095) overnight on a rotating wheel at 4°C. The samples were then incubated five times for 10 min with 1 ml of wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.9, 0.1% NP‐40, protease inhibitors) on a rotating wheel at 4°C. Immunoprecipitates were eluted in Laemmli buffer at 95°C for 10 min. Similar elution volumes and protein quantities, for the IPs and the inputs, respectively, were loaded on a gel. Western blots were performed using anti‐Sirt1 (Abcam, ab32441), anti‐IPO7 (Abcam, ab99273), anti‐GFP (Abcam, ab5450), anti‐KAP1 (Merck, MAB3662), HRP‐conjugated anti‐Flag antibody (Sigma, A8592), HRP‐conjugated anti‐HA antibody (Sigma, 12013819001), HRP‐conjugated anti‐goat antibody (Dakocytomation, P0449), HRP‐conjugated anti‐rabbit antibody (Santa Cruz, sc‐2004), and HRP‐conjugated anti‐mouse antibody (GE Healthcare, NA931V).