Quantitative Western Blotting

Retinas from Bbs5^–/– and wild-type (WT) mice (n = 5 per group) were homogenized in sample application buffer as described previously.²⁸ They were then separated by standard polyacrylamide gel electrophoresis, transferred to a supported nitrocellulose membrane (1212590; GVS North America, Sanford, ME, USA), and blocked for 1 hour at room temperature with 4% non-fat dry milk in TBST (20-mM Tris-Cl, pH 7.6; 0.1% [v/v] Tween 20), supplemented with 0.02% sodium azide. They were incubated overnight at 4°C in the same solution containing primary antibody (anti-BBS5²⁵ and β-actin antibody, 4967; Cell Signaling Technology, Danvers, MA, USA). Binding of secondary antibodies conjugated to horseradish peroxidase (62-6520, 31460; Thermo Fisher Scientific, Waltham, MA, USA) was detected using chemiluminescent reagents by exposure to film. For quantification, individual band intensity was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA). Bands were normalized to the β-actin loading control.