After overnight incubation at 4°C with the appropriate antibody, blots were washed 3 times with PBST.
When appropriate, blots were stripped with Reblot™ (Cat No 2060, Millipore).
Where appropriate, blots were stripped and reprobed for fibrillarin (sc-25397; Santa Cruz Biotechnology) or tubulin (T5168; Sigma Aldrich, St Louis, MO, USA).
Unfortunately, the antibodies which were suitable for immunofluorescence analysis did not show a good signal when used for immunobloting, and the appropriate antibodies for western blot detection were useless to perform immunofluorescence studies.
Protein extracts were prepared from freshly dissected brains and spinal cords at the appropriate stages and western blot analyses were performed as previously described.
Each of the three antibodies recognised a single protein band of the appropriate size on Western blotting.
Both antibodies recognized bands of the appropriate size on western blots and, with respect to the neuronal staining, yielded almost identical staining patterns in immunocytochemical experiments, which supports their specificity.
We selected 3 PI3K antibodies (class IA PI3K (p85), PI3K-C2α and PI3K-C2β) that recognised a protein of the appropriate size on Western blotting and identified their target in formalin-fixed paraffin embedded tissues.
GDH (cytosolic marker), CZ (lysosomal marker) and CRT (ER marker) were detected in the soluble fractions by Western blot using the appropriate antibodies.
The degradation product was then detected by Western Blot using the appropriate antibody followed by chemoluminescence and Quantification with the Image Quant software.
The protein fractions were separated via 10% SDS-PAGE and then analyzed via Western blot using the appropriate antisera described above.
