Quantity one software package

Mouse proteins (25 or 10 μg) in the membrane fraction were separated on precast NuPage 4-12% Bis-Tris Acetate gels (Invitrogen, Life Technologies) and transferred to a nitrocellulose membrane (Whatman protein nitrocellulose transfer membrane, 0.2 μm, Millipore). The membrane was blocked using 5% bovine serum albumin (Sigma-Aldrich) dissolved in 0.1% PBS-Tween 20 and then incubated at room temperature for 1 hour with agitation and blotted with the primary antibody overnight at 4°C. The subsequent day, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) and developed in a Versa Doc gel imaging system (Bio-Rad, Hercules, CA, USA). The Quantity One software package (Bio-Rad) was used for densitometry analysis. The protein concentrations were normalized to β-actin (1:1000).

NP and siRNA were mixed in 20 mM HEPES buffer (pH 7.4) for 30 min to allow formation of NP-siRNA complexes. Afterwards, a 1 mg/mL solution of CTX was thiolated through reaction with Traut’s reagent at a 1.2:1 molar ratio for 1 hr in the dark at room temperature. Concurrently, SIA was conjugated to amine functional groups on NP at 1 mg of SIA/mg iron in the dark with gentle rocking for 1 hr. Subsequently the thiolated CTX was reacted with the Iodoacetyl groups on the SIA at 1 mg CTX per 0.9 mg Fe, anchoring CTX to the surface of the siRNA-bound NP to form NP-siRNA-CTX.
To evaluate the degree of CTX attachment to NPs, NP-siRNA-CTX were prepared as described above after purification of unbound CTX through S-200 Sephacryl resin (GE Healthcare Life Sciences). Both purified and unpurified NP-siRNA-CTX were boiled in loading buffer containing 10% 2-mercaptoethanol. Released or unreacted CTX from both reduced and un-reduced NP-siRNA-CTX were separated from NP-siRNA-CTX through SDS-PAGE and quantified using the Quantity One software package (Bio-Rad) and a standard curve of CTX at known concentrations.