Ablation of Recycling Endosomes

From Ang et al. 2004, JCB 167: 531

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