Protein g plus protein a agarose suspension

HzAm1 cells were lysed in NP-40 cell lysis buffer, and total protein extracts (1 mg) were used for co-immunoprecipitation. The co-immunoprecipitation systems contained 25 μl of Protein G plus/Protein A-agarose suspension (Merck, USA) and 1 μg V5-tag antibody (Millipore, USA) or 1 μg Sirt2 antibody. The same amount of normal rabbit serum was used instead of antibodies as a negative control. Immunoblotting was performed with the corresponding antibodies, followed by incubation with Clean-blot IP (Thermo, USA) 1:1000, and subsequently, the blot was exposed to film.

MDA-MB-468 cells were treated with biotinylated BENDA (Bio-BENDA) in the presence or absence of an excess amount of free drugs for 4 h and then lysed in PhosphoSafe™ Extraction Reagent (Merck, Darmstadt, Germany). Lysate (200 μL) was precleared with a mixture of Protein G Plus/Protein A Agarose Suspension (EMD Millipore, Billerica, MA, USA) for 1 h at 4°C and protein A and G agarose were removed by centrifugation at 13,000 rpm for 10 min. The lysate (200 μL) was immunoprecipitated with STAT3 antibody (1 μL) overnight at 4°C on a shaker and the immunocomplex was captured by the addition of a Protein G Plus/Protein A Agarose Suspension (30 μL) slurry for 1 h at 4°C. Samples were washed five times with lysis buffer and then boiled four times in SDS-PAGE sample buffer and run on an SDS-PAGE gel. The protein was transferred to a nitrocellulose membrane. The membranes were blocked in 5% skim milk in TBS/0.1% Tween 20. They were then incubated with streptavidin-HRP diluted at 1:2000 in 1.5% skim milk in TBS (pH7.4)/0.1% Tween 20 overnight at 4°C. Immunoblots were developed by using a chemiluminescent substrate.

SGC7901 cells were lysed with Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing fresh protease inhibitors and PMSF. The lysates were then incubated with anti-p16 antibody (BD Pharmingen) overnight at 4 °C, followed by incubation with Protein G Plus/Protein A Agarose Suspension (Merck) for another 4 h at 4 °C. After washing 3 times with the ice-cold lysis buffer, proteins were released from the beads using SDS lysis buffer for 10 min at 95 °C and then resolved on 10 % SDS-PAGE gels and analyzed by immunoblotting. To block the nitrocellulose membranes, 5 % skimmed milk in TBST was used to reduce nonspecific background. Membranes were then incubated with primary antibodies overnight at 4 °C. After washing in TBST 3 times for 10 min each, membranes were incubated with secondary antibodies for 1 h at room temperature, and then washed again as before. Bound antibodies were detected using a chemiluminescence phototope-horseradish peroxidase kit according to the manufacturer’s instructions (Pierce, Rockford, IL, USA).