G box chemiluminescent imaging system

Immunoblotting was performed using standard techniques. Briefly, samples were run on 12% gels at 50–100V. Samples were then transferred overnight at 30V onto a PVDF membrane (Immobilon®-P, Millipore). The membrane was incubated for 1hr in 0.2% I-block reagent (Applied Biosystems), then incubated in 1:250 primary P-GSK antibody (cell signaling technologies #9336) over night at 4°C with gentle agitation. The Membrane was then rinsed 3 times with TTBS before incubation with 1:5000 secondary antibody (Jackson ImmunoResearch labs #111–035-003) for 1hr at RT. The membrane was then rinsed 3 times in TTBS and incubated for 5min in ECL (Western Lighting® ECL Pro, PerkinElmer) before being imaged using a G Box chemiluminescent imaging system (Syngene). The Membrane was then stripped via 20min incubation at 37°C with gentle agitation using Restore™ Western Blot Stripping Buffer (Fisher Scientific). The membrane was then rinsed 3 times in TTBS. The membrane was then probed and imaged again using similar procedures as described above, with 1:1000 GSK primary antibody (cell signaling technologies #9315) and 1:10,000 secondary antibody (Jackson ImmunoResearch labs #111–035-003). Densitometry was performed in FIJI/ImageJ with P-GSK normalized to total GSK.