The reprogrammed cells were harvested and lysed with cell lysis buffer (1% Triton X-100 in 50 mM Tris–HCl, pH 7.4 containing 150 mM NaCl, 2 mM Na3VO4, 100 mM NaF, and protease inhibitors). 10% of the cell lysate was used for Input. The rest of the lysate was incubated with antibody overnight at 4 °C and the target protein was captured by a 1:1 mixture of Ezview Red Protein A Affinity Gel (Sigma, P6486) and Ezview Red Protein GAffinity Gel (Sigma, E3403) for 4 h at 4 °C. The immunoprecipitated proteins were used to perform western blotting. The antibodies used in coimmunoprecipitation were anti-Sin3a (Abcam, ab3479), anti-Tet1 (Genetex, GTX124207), anti-HDAC1 (Santa Cruz Biotechnology, SC-8410), anti-Flag (CST, 14793s), and rabbit IgG (Millipore). rabbit IgG was used as a negative control.
Anti sin3a
Manufactured by Abcam
Anti-Sin3a is a lab equipment product that functions as an antibody. It is used for the detection and study of the Sin3a protein, which is a transcriptional regulator involved in various cellular processes.
Automatically generated - may contain errors
2 protocols using anti sin3a
1
Immunoprecipitation of Protein Complexes
2
Th17 Cell Immunofluorescence Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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T cells were cultured in Th17 conditions and treated or not with PMA and Ionomycin for 4 h. Cells were then deposited on Poly‐L‐lysine coated coverslips in duplicates, fixed for 10 min at RT with 3.7% Paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO4) and permeabilized with ice‐cold Sucrose‐HEPES‐Triton‐X100 buffer, for 5 min on ice. Coverslips were washed, blocked in 4% BSA, 10% Normal Goat Serum and eventually incubated with primary antibodies (1:200) for 1–2 h at RT. Anti‐ROR gamma (t) (AFKJS‐9), eBioscience, #14‐6988‐82 and anti‐Sin3A (Abcam #129087) were validated and used. After washing, coverslips were incubated with Alexa‐labeled secondary antibodies (1 μg/ml, Abcam), nuclei stained with Hoechst 33342, and images were sequentially acquired with a 63× 1.4NA objectives using Leica TCS SP5 confocal microscope equipped with 405, 488, 546, and 633 nm lasers obtaining images at 1024 × 1024 pixel resolution with 16‐bit pixel depth. Images were acquired and analyzed using ImageJ (Schindelin et al, 2009 ), and data processed in Excel. Plots and statistical analyses were performed with Prism software.
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