Cell culture and oxygen-glucose deprivation (OGD) treatment
Human embryonal teratocarcinoma Atera2/D1 (NT2) cells (Stratagene, La Jolla, CA), mouse Neuro-2a (N2a) neuroblastoma cells (ATCC CCL-131) and human HEK 293 cells  were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Bethesda, MD) supplemented with 10% fetal calf serum (GCS, Wisent, Inc. St. Bruno, PQ). NT2 cells were differentiated into neurons and astrocytes with all trans-retinoic acid (RA, Sigma, Oakville, ON) according to the method of Pleasure and Lee  as described previously . Mouse primary cortical neurons were prepared from embryonic E15-16 CD1 mice and cultured in neurobasal media for 5-14 days as previously described .
For OGD treatment, NT2 neurons and mouse primary neurons were washed once with glucose-free DMEM, and incubated in glucose-free DMEM with 10% FBS for 2 h in a hypoxic chamber (Forma 1025 Anaerobic Chamber; ThermoForma, Marietta, OH, USA). At the end of the OGD treatment, cells were removed from the chamber and returned to the incubator for 16 h. The same OGD treatment was performed with undifferentiated NT2 and N2a cells except the incubation in OGD conditions was increased to 6 h due to their resistance to OGD treatment. Cell viability for all cell lines was assessed by the Trypan Blue (Sigma, Oakville, ON) exclusion assay. In this procedure, all floating cells were collected from culture media and washing buffer, and then combined with the trypsinized cells. Cells were incubated in the Trypan Blue dye for 5 min. Labelled cells were counted using a hemocytometer.
RNA extraction, real time quantitative RT-PCR (qRT-PCR) and semi-quantitative RT-RCR
RNA extraction, first strand cDNA synthesis, and qRT-PCR analysis were performed as described previously . RNA pools extracted from frontal cortex of postmortem human brain samples described previously  were used for subtractive hybridization and qRT-PCR. Additional brain samples were obtained from the Human Brain and Spinal Fluid Resource Center (, VAMC, Los Angeles, CA), which is sponsored by NINDS/NIMN, National Multiple Sclerosis Society, VA Greater Los Angeles Healthcare System, and Veterans Health Services and Research Administration, Department of Veteran Affairs. To detect the expression level of the SDIM1 transcript in brain tissue and cultured cells, equal amounts of cDNA (2 ng each) were used with the primers: SDIM1F 5' GGGCCATGAACACATCACTTG 3' and SDIM1R 5' TCAGGTCAAAGTTGGCAATGAA 3'. The DNAJB4 transcript was detect by using the primers: DNAJB4F 5' TCTCAAACAAGACCCTCCCA 3' and DNAJB4R 5' ATGGCAGCCCATATCCAATA 3'. PCR was performed using a ABI 7500 FAST Real Time PCR system and reagents according to the manufacturer's instructions. The primers used for semi-quantitative RT-PCR for SDIM1 are: F 5' TCGGTGGGAAGACTGCTTAT 3' and R 5' CGTCTGTCCTGTGACATGGT 3'; for DNAJB4 are: F 5' TGGTTGTACCAAACGGATGA 3' and R 5' ATGGCAGCCCATATCCAATA 3'.
Plasmids, transient transfections and staining
Human cDNA encoding the mature (without the signal peptide) SDIM1 protein was cloned into the pGEX -3X vector (GE Healthcare, Baie d'Urfe Quebec) for GST-SDIM1 fusion protein production. The first amino acid after the signal peptide (position 27) was mutated from "C" to "A" in order to increase the solubility of the recombinant SDIM1 protein in E. coli. Human cDNA encoding full length SDIM1 protein was cloned in the pEGFP-N1 vector (Clontech, Palo Alto, CA, USA) with or without a stop codon added between the C-terminus of SDIM1 and the EGFP sequence (Invitrogen, Burlington, ON) to produce pSDIM1*EGFP, pSDIM1-EGFP respectively. The coding region of the human DNAJB4 cDNA was cloned into the pCMV-Tag1 vector to create a pCMV-DNAJB4-Tag1 construct.
For overexpression analysis, N2a cells were plated in 6-well plates at a density of 0.5 × 106 cells/well, 24 h before transfection. Cells were transfected with 5 μg pCMV-DNAJB4-Tag1 plasmid or pSDIM1*EGFP plasmid and 15 μl lipofectAmine 2000 reagent, or co-transfected with 2.5 μg each of the pSDIM1*EGFP and pCMV-DNAJB4-Tag1 plasmids plus 15 μl lipofectAmine 2000 reagent. Cells were collected for Trypan Blue exclusion assay as well as total RNA and protein extraction 16-24 h after transfection; or treated with OGD for 6 h plus 16 h recovery prior to Trypan blue assay or total RNA and protein extraction. For siRNA silencing, the siRNAs were purchased from GenePgarma (Shanghai GenePgarma Co, Ltd, Shanghai, PRC). Undifferentiated NT2 cells were plated in 12-well plates at a density of 0.25 × 106 cells/well, 24 h before transfection. Cell were transfected with 100 μM mixed human SDIM1 siRNAs containing a pool of two sequences: 5' UUAAACAGAGAUAUAAGUC 3' and 5' UUUAAUAGACCACAAACUC 3' and/or 100 μM mixed human DNAJB4 siRNAs containing three sequences: 5' UUGGAUAGUCUAGCACUUC 3', 5' UUUCUUCAGAAUCUCUACC 3' and 5' UUUCGAGAAAUCUUCAUCC 3' using Dharmafect1 transfection reagent according the manufacturer's instructions (Dharmacon, Thermo Fisher Scientific, Inc). Cells were subjected to 6 h OGD treatment 24 h after transfection and collected for Trypan Blue exclusion assay 16 h after re-oxygenation. For co-immunoprecipitation analysis, HEK 293 cells were plated in 10 cm plates at a density of 2 × 106 cells /plate, 24 h before transfection. Cells were transfected with 15 μg of pEGFPN1 or pSDIM1-EGFP alone, or co-transfected with 7.5 μg each of pSDIM1-EGFP and pCMV-DNAJB4-Tag1 plasmids DNA mixed with 45 μl LipofectAmine 2000 reagent. Cells were collected for total protein extraction 21 h after transfection. For cellular localization of SDIM1, mouse primary cortical neurons were stained (or double-stained) with anti-SDIM1 (dilution1:100 v/v), anti-MAP2 (1:200 v/v dilution, Novus Biologicals, Inc Littleton, CO), or anti-DNAJB4 (1:100 v/v dilution, abcam, Cambridge, MA) antibodies, followed by FITC -conjugated anti-rabbit IgG, rhodomine-conjugated anti-mouse IgG (for MAP2 and DNAJB4). The nuclei were counterstained with DAPI in PBS for 5 min and then mounted in Vectashield mounting medium (Vector laboratories, Burlingame CA, USA). The cells were viewed with a Zeiss Axiovert 200 M fluorescence microscope equipped with a Zeiss AxioCam camera (Zeiss, Midland, ON). The images were captured and analyzed using Zeiss Axiovision 3.1 software.
Antibody production and purification
Custom polyclonal antibody (GenScprit, Piscataway, NJ) was produced using synthetic peptide N'-LGSPLSLWSIKTPS. The immune serum was purified by immunoaffinity purification using recombinant GST-SDIM1 fusion protein. Briefly, purified GST-SDIM1 fusion protein was separated by SDS-PAGE and electro-blotted onto a nitrocellulose membrane. The Ponceau stained membrane portion containing the SDIM1 antigen was excised and subjected to a Western blotting procedure using 2 mL original crude serum. The bound antigen-specific antibody was eluted with 0.1 M Glycine-HCl buffer, pH 2.7. The eluted antibody was neutralized by adding 1/10 volume of 1 M Tris, pH 8.5, concentrated using Amicon Ultra-15 Centrifugal Filter Device (Millipore, Fisher Scientific, Ottawa, ON).
Protein extraction, Western blotting and co-immunoprecipitation
Recombinant GST-SDIM1 fusion protein was purified from Rosetta cells using a glutathione column according to the manufacturer's instruction (GE Healthcare, Baie d'Urfe Quebec). Mouse tissues were frozen in liquid nitrogen and homogenized in RIPA buffer in an electronic homogenizer and then kept on ice for 45 min. The samples were centrifuged at 14,000 xg for 10 min at 4°C to collect the supernatant for total cellular proteins. For total protein extraction from cultured cells, cells were trypsinized and collected by centrifugation. They were washed twice with PBS and lysed with RIPA buffer containing 1X protease Inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The lysate was vortexed and incubated on ice for 15 min, followed by sonication for 30 sec. In some cases the total cellular protein was freeze-dried and reconstituted in PBS in order to achieve a higher concentration. Western blotting analyses were performed as previously described . Human tissue protein blot was purchased from BioChain Institute, Inc (Hayward, CA, USA). Each lane contains 50 μg of total cellular protein. The blots were probed with the following primary antibodies: Rabbit polyclonal, affinity-purified anti-SDIM1 (1:5000), rabbit polyclonal anti-flag (1:1000, Rockland, Gilbertsville, PA), mouse monoclonal anti-DNAJB4 (1:1000, Abcam, Cambridge, MA), mouse monoclonal anti-EGFP (1:1000, Milipore, Temecula, CA), goat polyclonal anti-GST (1:1000, Amersham Phamacia Biotech, Baie d'Urfe, QC), mouse monoclonal anti-ubiquitin (1:1000) and mouse monoclonal anti-β-actin (1:5000, both Sigma, Oakville, ON), and mouse monoclonal anti-GAPDH (1:10,000 v/v, Milipore, Temecula, CA). The antigens were detected using horseradish peroxidase-conjugated secondary antibodies: anti-mouse IgG (1:5000 v/v), anti-rabbit IgG (1:5000 v/v, both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-goat IgG (1:5000, Sigma, Oakville, ON). The antigen-antibody complexes were visualized by enhanced chemiluminescence using an ECL Plus detection kit (Amersham Phamacia Biotech, Baie d'Urfe, QC).
For the co-immmunoprecipitation assay, flag-tagged DNAJB4 and EGFP-tagged SDIM1 constructs were transiently co-transfected into HEK-293 cells and total cellular proteins were extracted as described above. In another case, purified GST-SDIM1 protein was mixed with cellular proteins extracted from HEK-293 cells transfected with pEGFP-N1 or pSDIM1-EGFP. The immunoprecipitation procedure was as described previously  and precipitated complexes were boiled in protein loading buffer and separated by SDS-PAGE. The presence of SDIM1, DNAJB4, GST-SDIM1, and SDIM1-EGFP in the complex was revealed by Western blotting as described above.
Yeast two-hybrid screening
Human cDNA encoding the full length SDIM1 protein was cloned into the pGBKT7 vector (Clontch, Palo Alto, CA, USA) to generate a chimaeric open reading frame encoding the Gal4 DNA binding domain and SDIM1 protein. This construct was introduced into Saccharomyces cerevisiae strain AH109. A single colony containing cells harboring the pGBKT7-SDIM1 plasmid was then used to provide host cells for screening a human brain cDNA expression library constructed using the pACT2 vector (Clontech, Palo Alto, CA, USA). The protein-protein interaction was first screened by plating the transformants onto SD/-Trp-Leu-His-Ade selection plates. Positive clones were then re-screened for the presence of β-galactosidase activity to eliminate false interactions. Library plasmids harboring SDIM1 interacting proteins were rescued and re-introduced into the pGBKT7/SDIM1-containing host cells to further eliminate false interactions. The identity of the cDNA encoding SDIM1-interacting protein was revealed by DNA sequencing and database searches.