Las 3000 imager

Western blot was performed as described previously [10 (link)]. Briefly, cell lysates (30–50 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS PAGE) and then subsequently transferred to pure nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA. Blots were visualized with a chemiluminescence ECL detection system (EMD Millipore, Rockford, IL, USA) and analyzed using a FujiFilm LAS-3000 imager (Fujifilm, Tokyo, Japan). HRP-conjugated sheep anti-mouse, donkey anti-rabbit secondary antibodies were from GE Healthcare UK Limited (Pittsburgh, PA, USA). PXDN antibody was from Abnova (Walnut, CA, USA). Rabbit polyclonal phospho-p53 and Bax antibodies were from Cell Signaling Technology (Danvers, MA, USA), while mouse monoclonal total p53 was from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse monoclonal β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA).

Whole spinal cord protein samples from α₂^+/G301R and α₂^+/+ littermate mice exposed to SCI and allowed 3 days survival in addition to naïve α₂^+/G301R and α₂^+/+ littermate mice were prepared as described [50 (link)]. Equal amounts of protein were separated by SDS-PAGE on 10–14% (α_1, α₂, α₃ and AQP4) gels and electro-blotted onto nitrocellulose membranes (Pharmacia-Amersham). Membranes were blocked in PBS with 5% skimmed milk and 0.5% Tween-20 and incubated with the following primary antibodies: anti-α₁ diluted 1:2000 (clone a6f-c, Developmenal Studies Hybridoma Bank), anti-α₂ diluted 1:1000 (Merck Millipore), anti-α₃ diluted 1:1000 (Merck Millipore), anti-AQP4 diluted 1:1000 (AQP-004, Alomone labs) anti-GAPDH diluted 1:1000 (Abcam), or anti-β-Actin diluted 1:2000 (Sigma-Aldrich) overnight at 4 °C.
Next, membranes were incubated with HRP-conjugated secondary antibodies (swine anti-rabbit HRP diluted 1:2000 (Dako) or rabbit anti-mouse HRP diluted 1:2000 (Dako)) for 1 h at room temperature. Visualization of blots was done in a LAS 3000 imager (Fujifilm) with Amersham ECL Western Blotting Detection Kit (GE Healthcare). Post densitometric analysis and image processing of blots were performed in Image J.

Cells were lysed in radioimmunoprecipitation assay buffer supplemented with Complete Phosphatase and Complete Protease inhibitor cocktails (Roche). After dilution in Laemmli buffer with DTT and boiled for 5 minutes, samples were loaded on 4% to 20% Mini-PROTEAN TGX Precast Protein Gels (Biorad). Proteins were transferred on polyvinylidene fluoride (PVDF) membranes using a Transblot Turbo System (Biorad), blocked in 5% nonfat dry milk/PBS, and incubated overnight at 4 °C with primary antibodies. After washing, membranes were incubated for 1 hour with secondary antibodies (Abcam). Images were acquired using a Fujifilm LAS 3000 Imager. Densitometric analysis were performed with ImageJ software (Fiji, RRID:SCR_002285). Band signal intensity was normalized for the respective loading control values (actin or SDHA).
Primary antibodies: hypoxia-inducible factor 1 alpha HIF-1a antibody (Novus Cat# NB100-479SS, RRID:AB_790147), HIF-2a antibody (Abcam Cat# ab199, RRID:AB_302739), SDHA antibody (Abcam Cat# ab14715, RRID:AB_301433), 6X His-tag antibody (Abcam Cat# ab9108, RRID:AB_307016), beta Actin antibody (Abcam Cat# ab75186, RRID:AB_1280759). Secondary antibodies: Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341), Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307).

Mechanical dissociation with Accutase (ThermoFisher) was used to prepare single cell suspensions. Cells were counted using a hemocytometer. For colony formation assay, 350 cells were seeded in 5 cm dishes coated with polyornithine (Sigma) and laminin (Biolamina). U3082MG, U3084MG and U3065MG cells were cultured for 14 days while PIGPCs for 8 days, under the indicated conditions, then washed in PBS and fixed using 4% paraformaldehyde. Cells were stained using 0,01% crystal violet/H2O. Wells were washed gently in water, then air-dried for 24 hours. Images were acquired with a Fujifilm LAS 3000 Imager.
Sphere formation assay was performed with the hanging-drop method. 10 cells in 35 µL drops were seeded on the lid of a 48 well plate and grown under the indicated conditions for 2 weeks. For secondary sphere assay, primary spheres were pooled, pelleted, dissociated with Accutase and reseeded at the indicated conditions. Wells with spheres were manually counted and images were acquired with a Zeiss AX10 inverted microscope.

The promoter sequence of AnnAt1 was amplified by PCR using the primers 5′-AAGCTTAAACTGAACTTCTCCATCAATTTC-3′ and 5′-GGATCCCTTCTACTTTTAGTGTTTTGTGTATG-3′. The resulting fragment was cloned into the BamHI and HindⅢ sites of the vector pGWB535 to generate a fusion construct with luciferase (LUC). This vector was introduced into A. tumefaciens strain GV3101. For infiltration of Nicotiana benthamiana, the P19 protein of tomato bushy stunt virus was used to suppress gene silencing (Voinnet et al., 2003 (link)). Co-infiltration of A. tumefaciens strains containing the pAnnAT1::LUC (pA::LUC) construct and the P19 silencing plasmid with or without the 35S::AtPP2-B11 construct was infiltrated into leaves of 4-week-old N. benthamiana plants as described previously (Walter et al., 2004 (link)). For luminescence imaging, the plants were sprayed with 1mM d-luciferin (Wako, Japan) containing 0.01% Triton X-100. After 5min in the dark, the luminescence was observed using an LAS3000 imager (Fujifilm, Japan) and an LAS4000 imager (GE Healthcare, Japan).

HeLa cells were transfected with FLAG-Parkin C431S variants using Lipofectamine 2000 according to manufacturer's protocol and medium was replaced 4 h later. The next day, cells were treated with 10 µM CCCP for 0, 1, 2, 4, or 16 h. Cells were harvested in preheated (95°C) SDS lysis buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1%SDS). Lysates were homogenized by 10 strokes through a 23G needle. Protein concentration was determined by use of bicinchoninic acid (Pierce Biotechnology). To verify the band shift by oxyester formation, aliquots of lysates were treated with or without NaOH (100 mM final) for 1 h at 37°C. NaOH was neutralized by addition of equal amounts of HCl before samples were run on 8–16% Tris-Glycine gels and transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were incubated with anti-FLAG antibody (1∶100,000, Sigma F3165) overnight at 4°C followed by HRP-conjugated anti-mouse secondary antibodies (1∶15,000; Jackson ImmunoResearch 115-035-003). Bands were visualized with ImmobilonWestern Chemiluminescent HRP Substrate (Millipore) using a LAS-3000 Imager (Fuji).