Quantitation of the labelled profile during energy-dependent experiments and temperature-dependent experiments

(A, B) For energy controls, protoplasts were pretreated and incubated with different concentrations of sodium azide. (A) At low sodium azide concentrations uptake of nanogold was not inhibited but differences in the distribution of the probe were observed with respect to the control. (B) At high concentrations of sodium azide entry of probe was inhibited. (C) For low temperature experiments, protoplasts were incubated at 4 °C for 30 min before addition of positively charged nanogold and samples were taken after incubation with the probe for 45 min at 4 °C. An increase in the number of particles was observed on the PM with respect to the control and a corresponding decrease of probe was observed in peripheral and large vesicles. (D) Recovery of intracellular trafficking after incubation at low temperature was performed by bringing to 25 °C and taking samples after 15 min and 45 min. Quantitation data suggested that, during recovery, internalization was not reactivated while the recycling pathways was restored. The increase of the probe in inner vesicles and in vacuoles was due to the recovery of vesicle trafficking. (E, F) Effect of sodium azide and low temperature on Golgi and ER labelling. Sodium azide and cold experiments showed that a very low level of labelling was observed in Golgi bodies and ER (E) and a low percentage of Golgi bodies were labelled (F) compared to the control. Standard error (SE) is shown for each experiments.<p><b>Copyright information:</b></p><p>Taken from "Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold"</p><p></p><p>Journal of Experimental Botany 2008;59(11):3051-3068.</p><p>Published online 4 Jul 2008</p><p>PMCID:PMC2504345.</p><p>