The heavy standard in turn served to deduce absolute amounts of the endogenous proteins in the brain extracts using target protein-specific peptides.
To rule out that STZ administration may have altered expression level or pattern of transgenic tau, we performed immunohistochemistry of brain sections and Western blotting of brain extracts, using phosphorylation-independent tau specific antibodies, HT7 and TAU5.
A more expansive huntingtin (htt) interactome subnetwork, comprising over 700 candidate proteins, was likewise identified in mouse brain extracts using AP-MS by Shirasaki et al. [ 58].
We were unable to confidently identify SphK2 protein by western blotting on our brain extracts, using primary antibodies from a number of different suppliers.
Likely contributing to often contradictory and conflicting autoantibody results has been the use of immunoassay methodologies measuring autoantibodies against undefined antigens such as by immunohistochemistry of brain tissue and Western blot of brain extracts using human serum [6], [7], [8], [9], [10].
D-2HG levels in the brain extracts used in our LC-MS measurements varied highly, a likely consequence of variations in tumor/stroma ratios in the extracts.
All cell culture experiments were blinded with respect to the patient-derived brain extracts used for transduction and posterior analysis.
To compare global parenchyma volumes between groups, and to determine intracranial volume (ICV) for normalisation of subcortical volumes, each image was brain extracted using FSL's brain extraction tool [Smith, 2002] and segmented into gray matter, white matter and CSF compartments using FSL's Automatic Segmentation Tool (FAST) [Zhang et al., 2001].
Since Munc18-1 is also able to bind in an alternative mode to the assembled SNARE complex [12], [13], [15], [32] we also tested the binding of Glu379 in an assay based on pull down of assembled SNARE complexes from a rat brain extract using GST-complexin.
