The following antibodies were used: rabbit anti-PER1 (described previously) (Fahrenkrug et al., 2006), mouse anti-V5 [SV5-PK1], rabbit anti-V5, rabbit anti-Flag, rabbit anti-Myc (Abcam, Cambridge, MA,USA), mouse anti-Flag (M2), mouse anti-γ-tubulin, mouse anti-α-actin (Sigma), mouse anti-HA.11 (Covance, Montreal, QC, Canada), mouse anti-Myc (9E10) (Upstate, Lake Placid NY.
A first hint for isoform-specific substrate conversion came from the analysis of the hypothalamic-pituitary-gonadal axis, regulated by pE-GnRH, and the hypothalamic-pituitary-thyroid axis, regulated by pE-TRH, in QC knockout mice (Schilling et al, 2011).
The primary antibodies used where: mouse anti-βIII tubulin (Covance, Laval, QC, Canada); mouse anti-cortactin 4F11 and rabbit anti GAP-43 (Millipore, Billerica, MA, USA); goat anti-Arp3 (Santa Cruz); rabbit anti-calpain-2 (Cell Signaling Technologies, Danvers, MA, USA); mouse anti-synaptophysin (Sigma Aldrich Canada, Ontario, Canada).
Secondly, we quantified intracellular versus secreted QC in mouse hypothalamic GT1-7 neurons and in primary neurons over defined cultivation periods.
For this purpose, different neuronal cell types were used, i.e. the QC expressing mouse hypothalamic neuronal cell line GT1-7 [ 32], kindly provided by I. Vorberg/H.
The antiserum 5407 (Probiodrug, Halle/S., Germany) generated a specific band at the molecular weight of isoQC (43 kDa) when testing homogenates of wild-type and QC knock-out mice, but not isoQC knock-out mice (not shown).
QC knock-out mice were generated on the basis of a classical homologous recombination approach.
To determine the specificity of this unexpectedly abundant isoQC labeling, brain tissue of wild-type, isoQC knock-out and QC knock-out mice was analyzed.
It is important to note that in brains from QC knock-out mice, the neuronal marker proteins Ucn-1, TH and ChAT were detected in EWN, LC and Nbm neurons, respectively.
For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-101094).
The labeling generated by the isoQC antiserum 3285 in wild-type mouse brain was completely absent in isoQC knock-out but not in QC knock-out mouse brain tissue demonstrating the specificity of this antiserum for isoQC (Fig. 1b).
