Input. Pulldown GST. GST-Ahi1

Transcription

1 A kd GST-Ahi1 +GST-Ahi1 kd GST GST-Ahi1 C kd Control Input Hap1A Hap1 Pulldown GST GST-Ahi1 Hap1A Hap1 Hap1A Hap1 Figure S1. (A) Ahi1 western blot showing that pre-absorption of anti-ahi1 with GST-Ahi1 (+GST-Ahi1) eliminates immunoreactivity of Ahi1 (arrow) in mouse brain extracts. () Coomassie blue-stained gel showing purified GST and GST fusion protein containing full-length mouse Ahi1 (GST-Ahi1) that were used for in vitro binding. (C) In vitro synthesized Hap1A and Hap1 were incubated with purified GST or GST-Ahi1. The bound proteins were pulled down by glutathioneconjugated beads and subjected to Western blotting with anti-hap1. Left panel shows the input for binding. Control is in vitro synthesized sample without HAP1 plasmids. Right panel is Western blot analysis of proteins bound to GST or GST-Ahi1. The amount of input for in vitro binding and bound proteins were analyzed.

2 A Hap1 green Single labeling with anti-hap1 red Nuclear Ahi1 red Single labeling with anti-ahi1 green Nuclear Double labeling of transfected Hap1A and Ahi1 Hap1A Ahi1 Merged Double labeling of transfected Hap1 and Ahi1 Hap1 Ahi1 Merged Figure S2. Specificity of antibodies to Hap1 and Ahi1. (A) The hypothalamic region of adult mice at 5 months of age was immunostained by anti-hap1 (upper panel) or anti-ahi1. The sections were then incubated with fluorescent-conjugated secondary antibodies and Hoechst dye for double labeling. Note that there is no fluorescence bleeding in signal labeling. () Double labeling of HEK293 cells (upper panel) or PC12 cells (lower panel) that expressed both transfected Ahi1 and Hap1A or Hap1. This double labeling revealed the specificity of each antibody to its antigen.

3 A Calbindin Hap1 Merged Hap1 Ahi1 Nuclear Interposed cerebellar nuclei Medial cerebellar nuclei Vestibular nuclei Figure S3. Expression of Hap1 and Ahi1 in the mouse cerebellum. (A) Low magnification graphs of the mouse cerebellum showing costaining of Purkinje cells with antibodies to calbindin and Hap1. () Immunofluorescent staining of the cerebellum of adult mouse brains showing that Hap1 (green) and Ahi1 (red) colocalize in the same cells in the interposed cerebellar nuclei (upper panel), medial cerebellar nuclei (middle panel), and vestibular nuclei (lower panel). The brain sections were also stained with Hoechst dye to reveal the cell dense granule layers.

4 A Calbindin NeuN Nuclear Dentate nucleus C Cell number per image ** rainstem Cell number per image * 0 Figure S4. Comparison of the cerebellum from postnatal wild type () and Hap1 knockout () mice at P3. (A) Immunofluorescent staining of Purkinje cells with anti-calbindin antibody revealing the loss of Purkinje cells in Hap1 knockout mice at P3 (let panel). Immunofluorescent staining of the cerebellum of P3 mice showing that lack of Hap1 reduces the density of NeuN-positive cerebelluar neurons as compared to wild type () mice (middle panel). Hoechst staining of cellular nuclei is also presented (right panel). () romodeoxyuridine (rdu) incorporation assay for neurogenesis showing a significant reduction of the rdupositive neurons in the cerebellum (upper image) and brainstem (lower image) in Hap1 null mice aged at P1 to P2. (D) The number of rdu-positive cells per image (10X) in the cerebellum (upper panel) and brainstem (lower panel) of wild type and Hap1 null pups. * P<0.05 and ** P<0.01 (n=4-5).

5 Infection with control adenovirus GFP HAP1 Merged Infection with adenoviral HAP1 sirna GFP HAP1 Merged Figure S5. Suppressing neurites of cultured rat brain stem neurons by Hap1 sirna. Cultured rat brain stem cells (DIV13) were infected by control adenoviral GFP or adenoviral Hap1 sirna vector that also independently expresses GFP. Arrows indicated viral infected cells that express GFP. The cells were also labeled by anti-hap1. Note that Hap1 sirna viral infection reduces the expression of Hap1 and neurites compared to control infected neurons.

6 Supplemental Methods In vitro binding. PRK vectors (1!g) expressing Hap1A and Hap1 were used for in vitro synthesis of Hap1A and Hap1 with the TnT Quick Coupled Transcription/Translation System (Promega). The synthesized proteins (10!l) were incubated with 20!l GST-Ahi1-linked beads in 500!l PS buffer at 4 C for 2 h and then pulled down by glutathione beads. The beads were subjected to Hap1 western blot analysis. PC12 cell culture. PC12 cells were cultured on poly-d-lysine coated coverslips in DMEM/F-12 medium (Invitrogen) supplemented with 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin, and 100!g/ml streptomycin. Sixteen hours later, NGF was added to a final concentration of 100 ng/ml, and cells were cultured for an additional 24 h. Cells ( ) transfected with Ahi1 or Hap1 were examined to determine the percentage with neurites longer than 2 body diameters. romodeoxyuridine (rdu) labeling. Wild type or Hap1 knockout mouse pups at postnatal day 1-2 were injected with rdu (50 mg/kg, i.p.). rains of these mice were isolated 5 hr after injection and cut to frozen sections (8 10!m) with a microtone. The brain slices were then stained with anti-rdu antibody. The numbers of rdu-positive cells were counted in randomly chosen fields (10X) in the deep cerebellar nuclei and brainstem. For each animal, counting was made in 4 adjacent sections, and 3 and mice were analyzed. Statistical significance was examined by Student's t-test.