. 2022 Mar 25;23(7):3590. doi: 10.3390/ijms23073590

Table 3.

Protocols of cargo synchronisation.

Protocol	State of Golgi	Steps of Cargo Synchronisation
	Complex	Accumulation of Cargo in ER Exit Sites and Emptying of Golgi	Duration of Cargo Pulse	Block of Delivery of Visible Cargoes to the Golgi	Beginning of Video Recording
1. Small pulse (40-15-40)
A. E-40-15-40-small (the mini-wave)	Empty	16 h at 40 °C	15 min at 15 °C	Shift back to 40 °C	2 min after shift to 40 °C
B. F-40-15-40-small (the mini-wave)	Full	16 h at 40 °C	15 min at 15 °C	Shift back to 40 °C	10 min after shift to 40 °C
2. Large pulse (40-15-40)
A. E-40-15-40-large (the maxi-wave)	Empty	16 h at 40 °C	2 h at 15 °C	Shift back to 40 °C	2 min after shift to 40 °C
B. F-40-15-40-large (the maxi-wave)	Full	16 h at 40 °C	2 h at 15 °C	Shift back to 40 °C	10 min after shift to 40 °C
3. Pulse (40-32-40)
A. E-40-32-40-small (the 40-32 mini-wave)	Empty	16 h at 40 °C	5 min at 32 °C	Shift back to 40 °C	2 min after shift to 40 °C
B. F-40-32-40-small	Full	16 h at 40 °C	5 min at 32 °C	Shift back to 40 °C	10 min after shift to 40 °C
4. Emptying–pulse (32[CHM]-15-32[CHM])
A. E-CHM-15-CHM	Empty	16 h at 40 °C, then CHM for 3 h at 32 °C	2 h at 15 °C (without CHM)	Re-addition of CHM, shift back to 32 °C	2 min after shift to 40 °C
B. F-CHM-15-CHM	Full	16 h at 40 °C, then CHM for 3 h at 32 °C	2 h at 15 °C (without CHM)	Re-addition of CHM, shift back to 32 °C	10 min after shift to 40 °C
5. ER accumulation–chase (40-32)
A. E-40-32 (iFRAP)	Empty	16 h at 40 °C	None	Bleaching of whole cell less the Golgi area	5 min after shift to 32 °C
B. E-40-32 (piFRAP)	Empty		3 min		5 min after shift to 32 °C
C. F-40-32 (iFRAP)	Full		None		30 min after shift to 32 °C
D. F-40-32 (piFRAP)	Full		3 min		30 min after shift to 32 °C
6. Steady-state iFRAP
A. Steady-state iFRAP	Full	16 h at 40 °C	None	Bleaching of whole cell less the Golgi area	Immediately after bleaching
B. Steady-state piFRAP	Full	16 h at 40 °C	3 min	Bleaching of whole cell less the Golgi area	Immediately after bleaching
7. Pulse–outcoming wave
A. F-40-32-40 outcoming	Full	16 h at 40 °C	30 min at 32 °C	Shift to 40 °C for 30 min	0, 2, 4, 6, 8, 10, 12 min after shift

ER/IC, endoplasmic reticulum/intermediate compartment; iFRAP, inverse fluorescence recovery after photobleaching; piFRAP, post- inverse fluorescence recovery after photobleaching.