P27kip1

The following antibodies were used: mouse monoclonal antibodies against phosphotyrosine (PY99, sc-7020, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and p27^Kip1 (BD Transduction laboratories, Heidelberg, Germany), rabbit monoclonal antibodies specific for pS10-p27^Kip1 (#ab62364, Abcam, Cambridge, UK) or pTyr361-HIPK2 (#PA5-13045, Thermo Scientific, Rockford, IL, USA) and polyclonal goat antibody against green fluorescent protein (GFP, Rockland Immunochemicals Inc., 600-101-215, Gilbertsville, PA, USA). A rat monoclonal HIPK2 antibody was kindly provided by M. Lienhardt Schmitz (Justus-Liebig-University, Giessen, Germany).

HEY cells were transfected with negative control and CDK5 siRNA and then fractionated with the use of a Nuclear and Cytoplasmic Extraction Kit (Promega, Madison, WI) according to the manufacturer’s instructions. Equal amounts of protein from the cytoplasmic and nuclear extracts (10 μg) were subjected to immunoblot analysis with antibodies against p21Cip1 (1:300), p27Kip1 (1:1500), PARP (1:1000 dilution; BD, Franklin Lakes, New Jersey), and a-tubulin (1:1000 dilution; Sigma, St. Louis, MO).

Cells were lysed for 1 h at 4 °C in Triton X-100 lysis buffer (PBS containing 1% Triton X-100, 25 mM Tris, pH 7.5, and 157 mM NaCl) supplemented with a cOmplete Mini protease inhibitor cocktail tablet (Roche, Basel, Switzerland) and 1 mM glycerol phosphate, 1 mM NaF, and 1 mM NaOrthoV. Debris was pelleted by spinning samples for 20 min at 12,000 rpm at 4 °C, and protein concentrations were determined by Bradford Assay (Bio-Rad, Hercules, CA, USA).
Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking for 1 h at room temperature in 5% milk or bovine serum albumin in TBS-T, membranes were incubated with 1:1,000 dilutions of the following primary antibodies: actin (Sigma, St. Louis, MO, USA), caspase 8, caspase 9, tubulin (Cell Signaling, Beverly, MA, USA), caspase 2 (EMD Millipore, Billerica, MA, USA), cytochrome C, and p27 Kip1 (BD, San Jose, CA, USA). Membranes were then washed 3× with TBS-T before being incubated with appropriate horseradish peroxidase conjugated secondary antibodies (mouse and rabbit: GE Healthcare, Buckinghamshire, England; rat: Cell Signaling, Beverly, MA, USA). Chemiluminescent visualization of bands was performed using a Kodak film developer (Rochester, NY, USA).

For Grx1-roGFP2 in vivo experiment, chicken embryos at HH development stage (HH stage) 12–14 were electroporated unilaterally with 4–6 μg μl⁻¹ Grx1-roGFP2 construct and analysed at HH stage 20-21 (ref. 25 (link)). Embryos were dissected in cold PBS containing 20 mM N-ethylmaleimide (NEM) to block oxidation and maintained in the same solution for 30 min at 4 °C. NEM was removed by rinsing embryos once with PBS followed by 4% paraformaldehyde fixation for 90 min at 4 °C. Embryos were washed with PBS and equilibrated in 30% sucrose before embedding and sectioning. Control embryos were treated with 20 mM H₂O₂ or 20 mM DTT at room temperature for 10 min before blocking with NEM. Effective preservation of the redox status of roGFP by NEM was verified in HEK293 cells (Supplementary Fig. 6). Cryostat sections (12.5 μm) were imaged directly for Grx1-roGFP2 or immunostained to detect neuronal progenitors (Sox2, Santa Cruz, 2c-17320), postmitotic neurons (p27Kip1, BD Biosciences, 610241), motor neuron progenitors (Olig2, kindly provided by Dr B. Novitch) and postmitotic motor neurons (Isl1/2, kindly provided by Dr T.M. Jessell) as previously described25 (link)48 (link). Cy3- or Cy5-conjugated secondary antibodies were from Jackson Immunoresearch (715-605-150; 711-605-152, 705-165-147, 706-165-148). Anti-PAM antibody was kindly provided by Dr Betty Eipper.

Western blotting was performed as described previously [16] (link). Membranes were probed with antibodies directed against p27^KIP1 (BD Transduction labs, Lexington, KY; #610241), alpha smooth muscle-actin (Abcam, Cambridge, United Kingdom, #ab5694), desmin (#ab32362), actin (Santa Cruz Biotechnology Inc, Santa Cruz, CA; SC#1616) and goat anti-RanGAP1 antibody [17] (link). Western blot bands were quantified using QuantityOne software (Bio-Rad Laboratories, Munich, Germany) by measuring the band intensity (Area×OD) for each group and normalizing to α-actin. The final results are expressed as percent changes by normalizing the data to the control values.