Ablation of Recycling Endosomes

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From Ang et al. 2004, JCB 167: 531

Contents


Stock solutions

Human Tfn-HRP
  • 0.5 mg/ml in H2O + 20% glycerol
Acid Wash
  • 150 mM NaCl
  • 20 mM citric acid

pH 5.0, adjust with NaOH

DAB solution

weigh freshly to make a 10 mg/ml in PBS solution -> Dilute 1:100 to make 0.1 mg/ml DAB in PBS working solution, PROTECT FROM LIGHT

Other

30% H2O2
1% BSA in PBS

Protocol

Tfn labeling
  • Serum starve VSVG-transfected cells in SF-DMEM 1h at 40 ºC
  • Tfn-HRP 0.01 mg/ml (1:50 from 0.5 mg/ml stock) 45 min 40 ºC in SF-DMEM
  • If fluorescent transferrin to be added: 50 ug/ml Tfn-Alexa (1:100 from 5 mg/ml stock) 45 min 40 ºC in SF-DMEM together with Tfn-HRP
  • Wash SF-DMEM
  • Chase 20 min 40 ºC
Ablation on ice
  1. Wash 2x ice-cold PBS
  2. Wash 2x 5 min with acid wash to remove surface-bound Tfn-HRP
  3. Wash 2x ice-cold PBS
  4. Incubate cells for 1 hr on ice in the dark with rocking as follows:
    • ABLATION: 0.1 mg/ml DAB + 0.025% H2O2 (8.4 µl 30% solution per 10 ml DAB in PBS)
    • CONTROL: 0.1 mg/ml DAB, no H2O2
  5. Wash 2x 1% BSA in PBS
Chase
  1. Add warm HPMI + 100 ug/ml cycloheximide, chase at 32 ºC 1.5 h
  2. Wash PBS, FIX
  3. VSVG trafficks to PM in control cells, in ablated cells remains in Golgi/RE
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