The samples were electroblotted to positively charged nylon membrane (Amersham Life Science, Buckinghamshire, UK) using a semi dry transfer cell (BioRad Laboratories, Richmond, CA, USA).
#Transfer and blocking: a. Transfer protein on the gel to a nitrocellulose membrane for 1 hr at 12 V using a semi-dry transfer apparatus, 1× transfer buffer, and blotting sheets.
Briefly, SDS polyacrylamide electrophoresis was performed using 8% gels followed by protein transfer using a semi-dry transfer machine.
Samples were then electrotransferred to PVDF (polyscreen transfer membrane, PerkinElmer, Waltham, MA, USA) using a semi-dry transfer apparatus (Trans-Blot® Turbo™ Transfer System, Bio-Rad).
The resolved bands on the gel were transferred onto a 0.2 µm pore size nitrocellulose membrane at 17 V for 50 min with transfer buffer using a semi-dry transfer apparatus (Bio-Rad Trans-Blot, California, USA).
Protein was transferred to PDVF using a semi-dry transfer and western blotting performed using either of the two EpoR antibodies at concentrations of 1/1,000 (R&D Systems, MAB307 mouse anti-human monoclonal or R&D Systems, AF-322-PB goat anti-human polyclonal).
Proteins were transferred to PVDF-Plus transfer membranes (Fisher Scientific, Pittsburgh, PA) for immunoblotting, using a semi-dry transfer apparatus (Owl Scientific, Woburn, MA) according to manufacturer's instructions.
The resolved gel was transferred to 0.45 μm nitrocellulose membrane (Bio-Rad) using a semi-dry transfer apparatus (Bio-Rad) for 30 minutes at 20 V. Transfer efficiency was analyzed using a Ponceau S (Sigma) stain.
Gels were transferred onto PVDF membrane using a semi-dry transfer system (iBlot, Invitrogen, Camarillo, CA, USA).
Thereafter, proteins were transferred onto nitrocellulose membranes using a semi-dry transfer apparatus (Bio-Rad, CA, USA).
