Hrp conjugated anti rabbit secondary antibody

Proteins were isolated from cell lines using TRI Reagent TM solution (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol. Protein samples were subjected to 12% SDS/PAGE and Western blot analysis (. The primary antibody used was rabbit anti-EREG (1:1000, Origene, Rockville, MD, USA). Secondary antibodies: HRP-conjugated anti-rabbit secondary antibody (1:2000, Dako, Glostrup, Denmark) and HRP-conjugated anti-mouse secondary antibody (1:1000, Dako, Glostrup, Denmark). For antigen detection, we used the enhanced chemiluminescence (ECL) EasySee Western Blot Kit.

Isolated core particles were electrophoresed on a 1 % native agarose gel and transferred to polyvinylidene fluoride (PVDF) membrane and then immunoblotted with a polyclonal rabbit anti-HBc antibody (1:1,000; Dako, Glostrup, Denmark), as described previously [43 (link)]. Bound antibody was detected with a horseradish-peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Dako) followed by enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ, USA). Total cell lysates were then subjected to SDS-PAGE and the resolved proteins transferred to a PVDF membrane. The membrane was then incubated with monoclonal mouse anti-tubulin (1:1,000; Calbiochem, San Diego, CA, USA), polyclonal rabbit anti-COX-2 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-extracellular signal-regulated kinase (ERK) and anti-p-ERK (1:1,000; Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-c-jun N-terminal kinase (JNK) and anti-p-JNK (1:1,000 Cell Signaling Technology), polyclonal rabbit anti-p38 and anti-p-p38 (1:1,000 Cell Signaling Technology), or polyclonal rabbit anti-luciferase (1:500; Santa Cruz Biotechnology) antibodies. Immunoreactive bands were visualized using a HRP-conjugated secondary antibody (Dako) followed by ECL. Relative band intensities were measured using the Fujifilm Image Gauge V4.0 program.

For western blotting, 30–75 μg of protein was loaded on 7.5 or 12% polyacrylamide mini-gels, subjected to SDS–PAGE and subsequently transferred onto PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated in primary antibodies against ROCK1 (1:500), ROCK2 (1:200) (Abcam, Cambridge, UK), pRhoA^Ser188 (1:1,000) and RhoA (1:1,000) (SantaCruz Biotechnology, CA, USA) at 4°C overnight [34 (link)]. Following incubation in an anti-rabbit HRP conjugated secondary antibody (Dako, Glostrup, Denmark; 1:2,500) for 1 h at room temperature, proteins were detected using the ECL detection method. Membranes were then re-probed with β-actin (Abcam, 1:1,000) or GAPDH (Abcam, 1:10,000), which served as a loading control. Bands were analysed by densitometry using Image Lab Software (Bio-Rad).

Proteins were extracted from embryonic testes or kidneys using a lysis buffer containing 10 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% SDS in 1X PBS and 0.5 mM PMSF. The extracted samples were loaded on BoltTM 4–12% Bis-Tris Plus gradient gel (Invitrogen) and electrophoretically transferred to PVDF membrane. The protein loading was analyzed by Panceau S staining. After blocking with Super Block T20 (TBS) Blocking buffer (Thermo), the membrane was probed with primary antibody anti-Utf1 from rabbit (1:500 dilution, a24273 ABCAM). The antibody was detected with anti-rabbit HRP-conjugated secondary antibody (Dako). WB for mouse histone H3 as loading control employed polyclonal rabbit anti mouse histone H3 antibodies (1:5000 dilution; ab1971 ABCAM).