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For LPS binding, membranes were then blocked by 2 wt% BSA in PBS, pH 7.4, for 1 h at RT and subsequently incubated with radiolabelled LPS (40 µg/mL; 0.13×106 cpm/µg) for 1 h at RT in PBS [32].

The membranes were then washed with TBST, submerged in binding buffer, and the secondary peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) antibody (Bio-Rad) or anti-rabbit IgG 'True Blot HRPP conjugated antibody (eBioscience) was added.

The membranes were then washed with TBS-T, and antibody binding visualised by enhanced chemiluminescence (Amersham Biosciences).

Membranes were then incubated with horse radish peroxidase (HRP) conjugated secondary antibodies and antibody binding was detected using enhanced chemiluminescence (NEN Life Science Products, Boston, MA, USA).

Membranes were then processed as described [41].

Membranes were then exposed to KODAK X-ray film.

Membranes were then washed in TBS-Tween.

The membranes were then imaged for chemiluminescence.

Antibody binding to the membranes was then revealed using the Pierce ECL Western Blotting Substrate Thermo Scientificc), and chemiluminescence was detected on film (GE Healthcare).

Nonspecific binding sites were blocked (5% milk in TBST) and the membrane was then probed with antiprolactin (1  μg ml−1) overnight.

The nitrocellulose membrane was then immersed in 5% skim milk to block nonspecific binding for 1 h at room temperature.