Monoclonal actin antibody

HepG2 Cells were lysed in 2× SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.05% bromophenol blue) and sonicated for 10 s to shear DNA. The whole-cell extracts were then electrophoresed through 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc) with a Mini PROTEANII transfer apparatus (Bio-Rad). The membranes were blocked and probed in TBS-T buffer (1× Tris-HCl 50 mM pH7.6, NaCl 150 mM, 0.2% Tween-20) containing 5% non-fat milk. Primary antibodies (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb #4668, Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb #4511, Cell Signaling Technology, Inc.) at 1:1000 in TBS-T were incubated with the membrane overnight at 4°C. β-actin was used as an internal control for normalization and detected with a monoclonal actin antibody diluted at 1:2500 (Sigma, St. Louis, MO). After washing with TBS-T, the membranes were then further incubated with horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (1:5000; Sigma), The immunoreactive bands were visualized by enhanced chemiluminescence reagents (Perkin-Elmer Life Science Products, Boston, MA), and densitometry scanning was performed with the densitometer (Molecular Dynamics).

Cell lysis was performed using 2x SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.05% bromophenol blue). Cell lysates were sonicated for 10 s to shear DNA. Cell extracts were then electrophoresed through 10% SDS-polyacrylamide gels and transferred to 0.2 µm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) with a Miniprotein II transferring apparatus (Bio-Rad). The non-specific interaction of membrane was blocked with 5% non-fat milk and probed in TBS-T buffer (1x TBS buffer, 0.2% Tween 20) containing. Monoclonal rabbit anti-AR (1:5000), was used to detect the androgen receptor (Abcam #133273), Glucocorticoid Receptor (D8H2) XP® Rabbit mAb #3660, ERα Antibody (F-10): sc-8002, BRD4 (E2A7X) Rabbit mAb #13440, Brd2 (D89B4) Rabbit mAb #5848, PARP (46D11) Rabbit mAb #9532, Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173 and a monoclonal actin antibody diluted 1:2500 (Sigma, St. Louis, MO) was used to detect β-actin as the internal control to normalize protein loading. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (1: 5,000; Sigma). Enhanced chemiluminescence reagents (Perkin-Elmer Life Science Products, Boston, MA) were used to visualize the immunoreactive bands and the densities of protein bands were scanned analyzed using ImageJ software from the NIH.

Total proteins were extracted in SUB buffer at different time points (containing 8M urea, 2% mercaptoethanol and 0.5% SDS), and electrophoresed in 12% SDS-PAGE gels. Proteins were blotted into nitrocellulose membrane and probed with specific antibodies against PABPN1 (1:2000) (Abcam, USA). Parallel samples were probed using monoclonal actin antibody (Chemicon International, USA) to confirm the equal loading of lysates between lanes. After incubation with specific secondary HRP-conjugated antibodies, the membranes were revealed using the western blot chemiluminescence reagent plus kit (NEN Life Sciences Products, Boston, MA, USA).
Quantification of the band intensity was performed using the ImageJ software. Values were normalized by β-actin. Quantification of PABPN1 protein level was determined by western blot using the ImageJ software, normalized to actin and presented as fold change relative to the level of PABPN1 in untransfected HEK293T cells.