<i>Pspc1</i> is expressed in the midbrain during development.

<p>(A-D’) Sagittal analysis of the expression pattern of <i>Pspc1</i> transcript (blue) in E12.5 wild-type (C57Bl/6J) mice. TH immunohistochemistry staining (brown) was taken along to mark the mdDA neuronal field. (E-I) At E14.5, <i>Pspc1</i> is expressed in the mdDA area, but also in the rest of the CNS, as revealed by an overview image (J). (E’-H’) High levels of Pspc1 transcript are observed in the brain, overlapping with the TH positive domain. (E”-G”) Pspc1 is co-expressed in most TH positive neurons.</p

Mascot search results of proteins from LMX1B IP purified excised protein bands.

<p>Mascot search results of proteins from LMX1B IP purified excised protein bands.</p

Mascot search results of proteins from LMX1B-2 IP purified excised protein bands.

<p>Mascot search results of proteins from LMX1B-2 IP purified excised protein bands.</p

Possible interaction between PSPC1 and PSF, and between PSF and LMX1B.

<p>(A) PSPC1 is detected in E14.5 midbrain neurons, and has an interaction with PSF in these cells. (B) An interaction with PSF is shown in HIS IP, PSPC1 IP and LMX1B IP, in MN9D cells overexpressing LMX1B-HIS. (C ) Again, PSF interacts with LMX1B, as shown in a HIS and LMX1B IP. LMX1B IP reveals clear pull-down of LMX1B-HIS, which also observed in the PSF IP, and in lower amount in the control IP. (D) PSF interacts with LMX1B in developing midbrain neurons. (E) Confirmation of the interaction of PSF with LMX1B(-HIS), and with NURR1. NURR1 IP reveals pull down of the protein, however this is not shown in HIS or LMX1B IP. Moreover, in a NURR1 IP, no LMX1B could be observed. <i>Ctrl, control IP with normal host serum; IP, immunoprecipitation; (-), MN9D cells transfected with control(empty) vector; (LMX1B-HIS) MN9D cells transfected with LMX1B-HIS overexpression vector.</i></p

Mascot search results of proteins from the Ni-NTA purification set-up.

<p>Mascot search results of proteins from the Ni-NTA purification set-up.</p

LMX1B co-immunoprecipitated protein identification.

<p>(A) IP against HIS, PSPC1 and LMX1B on LMX1B-HIS OE MN9D cell lysate, followed by separation on silver-stained SDS gel. The PSPC1 IP shows two protein bands that might represent both PSPC1 isoforms. Five protein bands of the LMX1B IP were excised and used for mass spectrometry analysis (arrows). In addition, the 42 kDa protein was taken along, as a positive control, and was later confirmed as LMX1B protein. (B) Second IP experiment against HIS, PSPC1 and LMX1B on LMX1B-HIS OE MN9D cell lysate, followed by separation on silver-stained (FireSilver kit, Proteome Factory, Berlin) SDS gel, confirming and improving the pattern as described in the first IP experiment (A). The PSPC1 IP shows two protein bands that likely represent both PSPC1 isoforms, and they were used for mass spectrometry analysis. Five differential protein bands of the LMX1B and HIS IP were excised and used mass spectrometry analysis (arrows). <i>Gt IgG Ctrl, goat IgG control; Rb IgG Ctrl, rabbit IgG control.</i></p

Mascot search results of proteins from HIS IP purified excised protein bands.

<p>Mascot search results of proteins from HIS IP purified excised protein bands.</p

Affinity purified <i>Lmx1b</i>-HIS proteins.

<p>(A) <i>Lmx1b</i>-HIS purified proteins from MN9D cells, by means of HIS tagged affinity purification via Ni-NTA agarose beads, followed by separation on silver-stained SDS gel. The left lane represents proteins purified from control transfected MN9D cells. The right lane shows purified proteins from LMX1B-HIS overexpressing MN9D cells. Asterisks mark an observed differential protein band. (B) Western blot validation of the LMX1B-HIS overexpression in MN9D cells, followed by successful purification of LMX1B-HIS protein. Lysate shows clear LMX1B-HIS overexpression. Supernatant reveals unbound LMX1B-HIS after Ni-NTA bead incubation. Protein is strongly bound to the beads, as shown by an extremely low amount of protein that was detected in the first washing-steps. Following successful elution, beads were heated and used for a second, thorough elution; a large amount of protein was detected that was not eluted in the first elution step. (C) Detailed image of the differential protein band in the LMX1B-HIS OE sample (asterisks). Overexpressed LMX1B-HIS protein (based on size) was detected as well (arrow). <i>Ctrl, purified protein from control transfected MN9D cells; LMX1B-mycHIS, purified protein from MN9D cells overexpressing LMX1B-HIS.</i></p

TH expression in E14.5 <i>Phox2b-LacZ/LacZ</i> embryonic brains compared to wild-type littermates.

<p>(<b>a–j</b>) TH protein expression in wild-type (<i>Wt/Wt</i>) and <i>Phox2b</i> mutant (<i>LacZ/LacZ</i>) littermates. Medially, the dorsal-caudal area where <i>Phox2b</i> normally is expressed, shows a decrease or loss of TH expression in the <i>Phox2b</i> mutant (boxed areas and (<b>a'–b',f'–g'</b>)). In addition, a subtle decrease of TH expressing neurons is shown in the rostral domain of the mdDA system (<b>c'–d',h'–i'</b>). <i>LacZ, Phox2b-LacZ mutant; Wt, wild-type; for an embryonic midbrain reference picture, see figure </i><i>1kk</i>.</p

Loss of TH expression in E12.5 <i>Phox2b-LacZ/LacZ</i> embryonic brains.

<p>(<b>a–f</b>) TH protein expression in E12.5 <i>Phox2b</i> wild-type and homozygous mutant (<i>LZ/LZ</i>) littermates. Mild loss of TH is observed in the dorsal-caudal midbrain (arrowhead). Dashed white lines represent the mid-hindbrain boundary. (<b>d'–f'</b>) bGAL expression in cells normally expressing <i>Phox2b</i>, rostrally and caudally of the MHB, plus overlays with TH (<b>d''–f''</b>). (<b>g–l</b>) TH expression in the heterozygous <i>Phox2b-LacZ</i> mutant (<i>LZ/Wt</i>), compared to homozygous <i>Phox2b</i> mutant (<i>LZ/LZ</i>) littermates (<b>m–r</b>). (<b>g'–l'</b>) Co-expression of bGAL and TH is shown in the heterozygous mutant, whereas TH expression is lower in bGAL positive cells in the homozygous mutant (<b>m'–r'</b>). (<b>g''–r''</b>) bGAL expressing neurons also express TH. In <i>Phox2b</i> mutant neurons (bGAL positive cells), the number of cells co-expressing TH protein appears lower, as demonstrated by decreased co-localization (m'',n'',r''). <i>LZ, Phox2b-LacZ mutant; Wt, wild-type; for an embryonic midbrain reference picture, see figure </i><i>1kk</i>.</p