Horseradish peroxidase hrp conjugated antibody

Cells were lysed in ice-cold RIPA cell buffer (Sigma) supplemented with protease inhibitors (Sigma). The proteins were separated with a 4–12% PAGE gel and electrotransferred onto a PVDF membrane. The membrane was probed with primary antibodies and subsequently detected by horseradish-peroxidase (HRP) conjugated antibodies (Santa Cruz). The primary antibodies were β-catenin (Santa Cruz), Flag antibody (Sigma), HIF1α (Santa Cruz) and HIF2α (Santa Cruz).

T cells were purified from peripheral blood using the Rosette Sep T cell purification kit (Stem Cell Technologies Inc., Vancouver, CA). Jurkat cells (clone E6-1) and 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). The pcDNA3.1-SRSF1-HA plasmid was a gift from Dr. James Manley (Columbia University, NY). The pGL2-zeta promoter-luciferase constructs were a gift from Dr. Barbara Rellahan [25 (link), 26 (link)] and pGL2-basic vector from Promega (Madison, WI). SRSF1 antibody was purchased from Life technologies (Carlsbad, CA), CD3ζ χλoνε 6B.10) and horseradish peroxidase (HRP)-conjugated antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO). SRSF1 specific siRNA and control siRNA were purchased from Qiagen (Valencia, CA)

The purity of the rAAV serotype was examined by SDS-PAGE. rAAV samples with 1010 viral genomes were boiled in the 5 x oading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 200 mM DTT, 0.2% bromophenol blue, and 20% glycerol) for 5 min, and loaded on a 10% polyacrylamide gel. The samples were separated by SDS-PAGE and analyzed via sequential Western blotting as described previously [70 (link),73 (link)]. Briefly, the samples were transferred to the nitrocellulose membrane (Amersham Protran 0.45 µm Membrane; Cytiva, MA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween (TBST) and incubated with the primary antibody overnight. The membrane was washed thrice in TBST and incubated with the secondary horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz, 1:3000) for one hour. Immunoreactive proteins were further detected by the ECL Western blotting substrate (Gibco, MD).

Immunoblot analyses were performed as described previously [11] . Cell lysates were prepared by adding SDS-PAGE sample solution to cells and boiled for 5 minutes. Proteins were separated by SDS-PAGE on polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked with 2% skim milk for 1 hour, followed by incubation with the first antibody overnight at 4°C. Horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology) was used as secondary antibody. The bands were detected by ECL system (GE Healthcare). Densitometric analyses of the detected bands were performed with Image-J software.
The first antibodies used for this study are as follows; anti-phospho-STAT3 (#9131), anti-phospho-ERK1/2 (#9101), anti-ERK1/2 (#9102), anti-phospho-JAK2 (#3771), anti-JAK2 (#3230) and anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology. Anti-STAT3 (sc-7179), anti-gp130 (sc-656), anti-phospho-JAK1 (sc-16773), anti-JAK1 (sc-7228), anti-LIF receptor (LIFR) (sc-659), anti-IL-11 receptor α (IL-11Rα) (sc-993) and anti-PTP1B (sc-1718) antibodies were obtained from Santa Cruz Biotechnology. Anti-GAPDH antibody (MAB374) was purchased from Millipore, and anti-Bip/GRP78 (610978) antibody was from BD Biosciences.

Proteins expressed in the sciatic nerve were determined by western blot analysis. Sciatic nerves were rapidly dissected from rats and used for protein analysis. All the procedures including protein extraction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and western blotting were essentially the same as described in our previous report [8 (link)]. Briefly, cells in the sciatic nerves were lysed using triton lysis buffer, sonicated, and centrifuged to collect the supernatant containing proteins. Protein (15 μg) was used for SDS-PAGE (10%) and western blotting analyses. Anti-phospho-Erk1/2 (1 : 1250, rabbit polyclonal, Cell Signaling, Seattle, USA), anti-p38 (1 : 1250, rabbit polyclonal, Santa Cruz Biotech.), anti-TNF-α (1 : 1250, rabbit polyclonal, Sigma), and anti-actin (1 : 10,000, mouse monoclonal, MP Biomedicals, Santa Ana, USA) antibodies were used as primary antibodies, and horseradish peroxidase- (HRP-) conjugated antibody (1 : 1250; goat anti-rabbit or goat anti-mouse, Santa Cruz Biotech.) as a secondary antibody. Protein band intensity in the scanned images of X-ray film was determined by using the i-Solution software (Image & Microscope Technology, Daejeon, Korea).