Ab10901

The acetylation of FOXP3 was detected using an IP kit (Absin, abs955-50 tests) according to the manufacturer's instructions. Briefly, the collected cells were washed with PBS and lysed with IP lysis buffer on ice for 5 min. Cells were scraped from the plate and transferred to a microcentrifuge tube. After ultrasonic disruption 3 times, the cells were subjected to centrifugation (14,000 g, 10 min) at 4°C, and the supernatant (cell lysate) was transferred to a new tube. Cell lysates (200–1000 µg total protein) were mixed with anti-FOXP3 antibody. After overnight incubation at 4°C, the protein A/G plus agarose was added to the sample and incubated on a rotator at 4°C for 2 hours. The mixture was centrifuged at 12,000 g for 1 minute to retain the precipitate, and it was washed with wash buffer. The acetylation of FOXP3 was determined by Western blotting with antiacetylated-lysine antibody (Cell Signaling Technology, 9941) and anti-FOXP3 antibody (Abcam, ab10901).

Cell lysates were abstracted using Thermo Scientific RIPA Buffer (Cat No. 89901) which contains 25 mmol/L Tris‐HCl (pH 7.6), 150 mmol/L NaCl, 1% Nonidet P 40 (NP‐40), 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Samples were centrifuged at 4°C, and the supernatants were collected. Quantified proteins were boiled with 5xloading buffer at 100°C for 5 minutes. Thirty micrograms protein was separated with 12% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS‐PAGE), and the separated protein was transferred to polyvinylidene fluoride (PVDF) membrane and then blocked in 5% nonfat milk medium under room temperature for about 2 hours. Primary antibodies Anti‐c‐Myc (1:1000, ab32072), Anti‐FOXP3 (1:1000, ab10901), Anti‐Bax (ab32503, 1:1000), Anti‐Bcl‐2 (1:500, ab59348), and Anti‐GAPDH (1:2500, ab9485) (Abcam, Cambridge, MA, USA) were added on the membranes and incubated overnight. After washed in Tris buffer saline Tween‐20 (TBST), HRP‐labeled goat anti‐rabbit secondary antibody IgG (1:2000, ab205718, Abcam) was added to the membranes, The mixture of primary and secondary antibodies was finally incubated for 1 hour and then washed with TBST for three times. Grayscale scanning was then used to visualize the immunoreactive proteins.

Total proteins were extracted by RIPA lysis buffer (Beyotime, P0013B), and the concentration of the proteins was measured by a BCA kit (Beyotime, P0012). Equal amounts of protein lysates were loaded on a sodium dodecyl sulfonate-polyacrylamide gel (SDS–PAGE) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat milk and incubated with antibodies at 4°C overnight. The primary antibodies used were as follows: anti-CD81 (1 : 1000, Cell Signaling Technology, 56039), anti-CD63 (1 : 1000, Abcam, ab68418), anti-CD9 (1 : 1000, Abcam, ab223052), anti-SIRT1 (1 : 1000, Abcam, ab263965), and anti-FOXP3 (1 : 2000, Abcam, ab10901). GAPDH was used as a loading control. Then, a horseradish peroxidase (HRP)-labeled secondary antibody was used to detect the specific protein bands.