All blots were washed with three changes of TBST for a total of 45 min.
The blots were washed with PBST and developed on Amersham Hyperfilm ECL (28906839, GE Healthcare, Little Chalfont, UK) using Amersham ECL Plus Western Blotting Detection Reagents (RPN2132, GE Healthcare).
Blots were washed with PBST for 10 min at RT, exposed to chemiluminescence reagent (Pierce, Milwaukee, WI, USA), and developed.
Blots were washed with TBST, and incubated in primary antibody against RIIβ for 2 hrs at room temp.
Next morning blots were washed with 0.05% Tween in PBS, and incubated with secondary antibody for 1 h.
In all subsequent incubation steps, the blots were washed with PBS-T alone or incubated in PBS-T containing antibodies.
Blots were washed with PBST (4×10 min each) at room temperature on a bench top shaker.
Antibodies (indicated in 'Materials') were added in TBS-T containing 2.5% skimmed milk and the blots were washed with TBST (TBS +0.1% Tween 20).
Blots were washed with TTBS twice for 15 min each and four times for 5 min each, and then developed with a homemade ECL chemiluminescence kit.
Subsequently, blots were washed with TBS containing 0.05% Tween 20 and incubated with the corresponding secondary antibody (see Table 3) diluted in blocking buffer.
After secondary antibody incubations, blots were washed with TTBS and developed by enhanced chemiluminescence using a standard kit (ECL, Amersham Pharmacia Biotech, Piscataway, NJ).
