Anti flag and anti β actin antibodies

H1299 and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO₂ at 37 °C. For mammalian expression of p32 and p53, the corresponding cDNAs were amplified by PCR and ligated into the correct reading frames of pIRESneo (Clontech Laboratories, Inc., Mountain View, CA, USA) containing 5′ FLAG or hemagglutinin (HA) coding sequences. For bacterial expression of p32, p53, SRp30c, and p66α, their cDNAs were amplified by PCR and inserted into pET15b and/or pGEX‐4T1 vectors. To generate mutant p32 expression vectors, wild‐type p32 cDNA was mutated by using Q5® Site‐Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) after the construction. All constructs were verified by DNA sequencing. Further details of plasmid constructions are available upon request. Antibodies used in this study are as follows: anti‐β‐actin and anti‐FLAG antibodies from Sigma‐Aldrich, St. Louis, MO, USA; anti‐His antibody from LifeTein, Somerset, NJ, USA; anti‐lamin antibody from Active Motif, Carlsbad, CA, USA; anti‐HA antibody from Proteintech, Rosemont, IL, USA; and anti‐tubulin, anti‐p32, and anti‐p53 (DO‐1) antibodies from Santa Cruz Biotechnology, Dallas, TX, USA.

For exogenous immunoprecipitation assays, HEK293T cells were plated in 60 mm dishes and then transfected with the indicated plasmids. After 24 h, cells were harvested using RIPA buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with mixed protease inhibitors. Cell lysates were incubated with 2 μg of the indicated antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight. Beads were eluted five times with lysis buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0, 165 mM NaCl, 0.5% NP-40), and thereafter, a western blot assay was performed to analyze the association by using the indicated antibodies. Western blot was performed as previously reported 56 (link). Anti-β-actin and anti-Flag antibodies were purchased from Sigma. Anti-Myc antibody was purchased from Santa Cruz Biotechnology. Antibody against CREPT was produced in the laboratory 57 (link).