Ab29170

Independent immunoblot analyses were performed on short-and long-term treated tissue, as previously described (Lum et al., 2016) (link). In brief, samples containing 5 µg of protein were resolved in 4-20% TGX precast gels (Bio-Rad, Australia), and subsequently transferred to a PVDF membrane (Bio-Rad). This loading concentration was determined to be in the linear range of a standard curve (Lum et al., 2017) . The membranes were incubated in the primary polyclonal antibodies at the following concentrations: anti-mGluR1α (1:15 000; DH510, Cell Signalling), anti-mGluR5 (1:5000; ab29170, Abcam), anti-Norbin (1:7500; Ab130507; Abcam), anti-Homer1a (1:5000; sc-8922, Santa Cruz) and anti-Homer1b/c (1:10 000; Ab97593; Abcam). Membranes were subsequently incubated with horseradish peroxide conjugated secondary antibodies. Bands were visualised using chemiluminescence detection reagents (GE Healthcare, Australia) and membranes exposed to Hyperfilm (GE Healthcare, Australia). Films were scanned using a GS-800 scanner (Bio-Rad) and densitometry values were quantified. Relative densitometry values for each protein were normalised to their respective β-actin levels and an internal control value (containing equal amounts of all samples), to account for protein loading and gelto-gel variability, respectively. Each sample was run in duplicate.