Immobilon western substrate

Rat colon tissues were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime, Haimen, China), and tissue lysates were centrifuged at 14,000 × g for 30 min. Supernatants were collected and mixed with 5× sodium dodecyl sulfate (SDS) sample buffer. The samples were separated by SDS-polyacrylamide gel electrophoresis using 8–12% acrylamide gels, and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following incubation with primary and secondary antibodies, protein bands were detected with Immobilon Western Substrate (Millipore) and analyzed using the Bioimage analysis system (Syngene, Frederick, MD, USA). The following antibodies were used: rabbit anti-ZO-1, rabbit anti-occludin (Cat.: ab31721; Abcam), rabbit anti-phospho-ERK, rabbit anti-ERK (Cat.: 4695; CST), rabbit anti-phospho-P38 (Cat.: 4511S; CST), rabbit anti-P38 (Cat.: 8690; CST), rabbit anti-phospho-c-Jun N-terminal kinase (JNK) (Cat.: 4668; CST), and rabbit anti-JNK (Cat.: 9252; CST).

SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD⁺, 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C. Nicotinamide (1 mM) was used as a positive control of SIRT2 inhibitors^{42 (link)}. The samples were analysed by 13.5% SDS/PAGE, stained with Coomassie Brilliant Blue R-250 containing 2-mercaptoethanol. The same samples were also electrotransferred onto Immobilon-P^SQ transfer membranes and subjected to Western blot. The blot was developed using a monoclonal mouse antibody against acetylated α-tubulin at Lys-40 (1:5000, clone 6-11B-1, Sigma T6793), antibody binding was revealed by anti-mouse IgG-peroxidase conjugate (Fc-specific), (1:5000, Sigma A2554). Peroxidase reaction was detected using Immobilon Western substrate (Millipore) by a Bio-Rad ChemiDoc MP Imaging system and its ImageLab 4.1 software. Then amido black solution (0.1% w/v amido black, 25% v/v isopropanol and 10% v/v acetic acid) was applied to stain the protein bands on the membrane for 1 min. Intensity of bands was analysed by ImageJ 1.49 using Measure command and subtracting background values. Relative acetyl-tubulin level corresponds to the difference measured in absence and the presence of 0.6 μM SIRT2.

Cells were washed in PBS buffer before being lysed with EBC buffer (50 mM Tris, pH 8.0; 120 mM NaCl; 0.5% NP-40) supplemented with a protease inhibitor cocktail. The same total amount of each lysate was loaded and resolved by SDS–PAGE and analyzed by immunoblotting. The blots were developed with Super Signal Pico substrate (Pierce Biotechnology, Rockford, IL) or Immobilon Western substrate (Millipore, Billerica, MA). The antibodies were the same as the ones used for IHC.

Cells were harvested by scraping with RIPA buffer. Extracts were incubated for 2 h at 4°C and obtained by centrifugation (13,000 rpm for 20 min at 4°C). Protein concentrations were determined using the BCA assay Kit (Thermostat, Hercules, CA, USA), and whole-cell extracts were adjusted to same amount of total protein (20 μg). Samples were electrophoresed in 10% SDS-PAGE. Proteins were then transferred onto a PVDF membrane (Millipore Corporation, Billerica, MA, USA) at 300 mA for 90 min at 4°C, and the membranes were incubated with 3% BSA (Sigma-Aldrich, St. Louis, MO, USA) in TBST to block nonspecific binding. Primary antibodies were incubated over night at 4°C. And we washed five times with TBST (0.5% tween 20 in 1x TBS) the secondary antibodies conjugated horseradish peroxidase (HRP) (Santa Cruz, Dallas, Texas, USA) that was applied and incubated for 1 h at RT. After five times of washing with TBST (0.5% tween 20 in 1x TBS), the membrane followed by chemiluminescent detection using Immobilon Western Substrate (Millipore Corporation, Billerica, MA, USA) with the ChemiDoc MP Imaging system (Bio-Rad Laboratories Inc., Hercules, California, USA). The antibodies diluents were shown at Table 1.

Differentiated human iPS cells were harvested after one month of culture in PI medium by scraping them into RIPA buffer (Thermo Fisher Scientific, San Jose, MA, USA). Cell lysates were incubated for 2 h at 4 °C and then centrifuged. Protein concentrations in the supernatants were determined using a BCA assay Kit (Thermostat, Hercules, CA, USA), and equal amounts of protein were separated by 10% SDS-PAGE (Whatman, Inc., Clifton, NJ, USA). The proteins were then transferred onto a PVDF membrane (Millipore Corporation, Billerica, MA, USA) and incubated with primary antibodies at 4 °C overnight. Primary antibodies were detected by adding secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Dallas, TX, USA) and incubating for 1 h. The membrane was then washed five times, and chemiluminescence was detected using the Immobilon Western substrate (Millipore Corporation, Billerica, MA, USA) and a ChemiDoc MP Imaging System (BioRad Laboratories Inc., Hercules, CA, USA). Antibodies and their dilutions are shown in Table 1.

Cells were extracted using a lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA (pH 8.0), 1% Triton X-100, 1 mM PMSF, 20 mg/ml aprotinin, 50 μg/ml leupeptin, 1 mM benzidine, 1 mg/ml pepstatin, 8 mM sodium pyrophosphate, and 20 mM β-glycerophosphate]. Forty micrograms of proteins were electrophoretically separated with 8–15% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking with TBS-T buffer [20 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] containing 5% skim milk, membranes were incubated at 4°C for overnight with primary antibodies (Runx-2, MMP-9, and β-actin; Cell Signaling Technology, Danvers, MA, USA). Membranes were washed with TBS-T buffer and then incubated at room temperature for 2 h with goat anti-mouse IgG or goat anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). The membranes were then washed with TBS-T buffer, visualized with Immobilon Western substrate (Millipore Corporation, Billerica, MA, USA), and detected with the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). The band density was then normalized to the β-actin reference.

Proteins were resolved by Mini Gel SDS-PAGE (Biorad^® system) and transferred to PVDF membrane (Immobilon-P^®- Millipore^®) according to standard procedures. Blocking and antibody incubations were performed in TBS - 0.2% Tween-20^® 5% milk (Marvel^®). The following primary antibodies were used: anti-Ku80, (1:1000)^{38 (link)}, anti-actin (1:5000, Santa-Cruz^® sc-1615), anti-H2AX-P (1:1000, Abcam® ab11174), anti-H3 (1:2000, Abcam^® ab12079), anti-flag (1:2000, SIGMA^® F1804 and F2425), anti-mono-ADPr AbD33205 (1:1000)^{34 (link)}, Anti-H3S10-p (1:1000, Bethyl^® A301-844A-T, and Abcam^® ab5176). Global ADP-ribosylation sigma was detected using the reagent anti-PAN-ADPr (1:2000, Merk^® MABE1016). Appropriate HRP-conjugated secondary antibodies were used anti-mouse (1;10000, DAKO^®), anti-rabbit (1;10000, DAKO^®), anti-goat (1;10000, DAKO^®) and anti-human (1:5000)^{34 (link)}. Immuno-reactive bands were detected by chemo-luminescence induced by Immobilon^® western substrate (Millipore^®), detected with the LI-COR^® Odyssey-Fc machine and quantified using Image-Studio^® software.