Supplemental Materials

This article contains the following supporting material:

• Figure S1 - CFP-Rab1bwt expressing cells were not visualized with excitation at 488 nm and emission recovered with a LP530 nm filter. This condition was used to quantify GFP fluorescence intensity in GFP-Arf1/CFP-Rab1b coexpressing cells.
• Figure S2 - RNAi treatment to knock-down Rab1b levels in HeLa cells performed after 72 h of transfection with 100 nM of either Rab1b or control siRNAs. (A) Measurement of Rab1b levels by Western blot quantification (three independent experiments). The graph shows averages of Rab1b density relative to calreticulin density (used as a loading control) calculated for each point. Rab1b Relative density in control was taken as 100%. Error bars represent standard deviation. (B-G) Subcellular localization of ß-COP and GM130 in either control or Rab1b silenced cells (indicated at left). Bars represent 10 μm. (H) Cells treated with Rab1b siRNA or control siRNA were homogenized and subjected to cell fractionation. Equivalent amounts of the PNS, cytosolic and membrane fractions were resolved by SDS-PAGE and Western blotted with anti-GBF1 antibodies. The same nitrocellulose filter was probed with anti-calnexin and anti-ERK1 antibodies to provide recovery standards for membranes and cytosol, respectively. The amount of GBF1 and calnexin was quantified by densitometry in PNS and membrane fractions. The graph shows averages of GBF1 density relative to calnexin density in PNS and membrane fractions from two independent experiments. GBF1 relative density in PNS was set as 100% in both control and Rab1b depleted cells. Error bars represent SD.
• Figure S3 - Quantification of Golgi intensity in GFP-Rab1bwt expressing cells (open circle) (n=5) and in GFP-Rab1Q67L expressing cells (filled circle) (n=4). Error bars represent SD.
• Video 1 - Time-lapse of GFP-Rab1bwt in HeLa cells. The movie shown contains stills from Figure7. Quantification of dynamic patterns is shown in Table1. Images were acquired every 4 s over a total time of 10 min using a Zeiss LSM Pascal confocal-scanning microscope. The movie was compressed into 7 fps and edited with ImageJ processing software (www.rsb.info.nih.gov/ij).
• Video 2 - Time-lapse of Sec13-YFP in HeLa cells. Quantification of dynamic patterns is shown in Table1. Images were acquired every 4 s over a total time of 10 min using a Zeiss LSM Pascal confocal-scanning microscope. The movie was compressed into 7 fps and edited with ImageJ processing software (www.rsb.info.nih.gov/ij).
• Video 3 - Time-lapse of p58-YFP in HeLa cells. Quantification of dynamic patterns is shown in Table1. Images were acquired every 4 s over a total time of 10 min using a Zeiss LSM Pascal confocal-scanning microscope. The movie was compressed into 7 fps and edited with ImageJ processing software (www.rsb.info.nih.gov/ij).
• Supplemental Table 1 - Quantification of dynamic patterns of peripheral structures labeled with GFP-Rab1b, Sec13-YFP or p58-YFP. Immobile structures were those that remained within a limited area (2 μm diameter) for at least 5 min. Mobile structures were those that traveled more than 2 μm from the starting point during a 5 min period, and transient structures were those that vanished or appeared during the same time period. Analysis of GFP-Rab1b labeled peripheral structures indicated that 23% of structures were immobile. Mobile structures represented 6% of the total, with some describing peripheral long-range randomly directed movements, while a few others emerged from the periphery and moved towards the Golgi. The majority of them (~70%) were transient, appearing or vanishing over a variable but short period of time (less than 5 min). COPII labeled structures were mostly immobile with a minimal proportion being transient or mobile. P58-YFP labeled structures were immobile (~9%), transient (~34%) or described a highly mobile measurable trajectory (~57%).