![Figure 5. RAB6 and KIF5B are required for the proper secretion SBP-EGFP-CD59, TNFα-SBP-EGFP, and SBP-EGFP-ColX at the plasma membrane; RAB6 depletion delays total protein secretion. (A) Left: HeLa-TNFα-SBP-EGFP, treated or not with RAB6 siRNAs, were incubated for 30 or 60 min with biotin to allow cargo release. The amount of secreted TNFα was determined on images acquired on cells using the SPI assay. Representative images of cells expressing TNFα-SBP-EGFP (green) and stained with anti-SBP (red) are displayed. Right: Quantification of the amount of TNFα present at the plasma membrane in cells treated as above (mean ± SEM, n = 67–111 cells). **, P < 10−2 (Student’s t test). Scale bars, 5 µm. (B) Left: HeLa-SBP-EGFP-ColX treated or not with RAB6 siRNAs were incubated for 30, 60, or 120 min with biotin to allow cargo release from the ER. The amount of secreted cargo was revealed by Western blotting using an anti-GFP antibody. β-tubulin was used as a loading control and as a nonsecreted protein. Representative immunoblots are displayed. Right: Quantification of the amount of ColX present at the plasma membrane in cells treated as indicated above (mean ± SEM, n = 3). *, P < 0.05 (Student’s t test). (C) HeLa cells expressing SBP-EGFP-CD59, TNFα-SBP-EGFP, and SBP-EGFP-ColX were treated for 3 d with control, RAB6, or KIF5B siRNAs. Cells were incubated for 30 min with biotin to allow cargo release from the ER. Cells were imaged using a time-lapse spinning disk confocal microscope. Vesicle velocity (µm/s) and the number of vesicles per cell were quantified using ImageJ (mean ± SEM, n = 17–35 cells). *, P < 0.05; **, P < 0.005; ***, P < 10−4 (Student’s t test). (D) The SUnSET assay was used to determine the effect of RAB6 depletion on total protein secretion. MEFs prepared from RAB6 loxP/Ko Rosa26CreERT2-TG embryos (described in Bardin et al. [2015]; named after MEF RAB6lox/lox) were treated with ethanol or with 4-OHT for 96 h to induce RAB6 depletion. Cells were then incubated with puromycin and chased in puromycin-free medium for 0, 1, 2, 4, or 5.5 h. Total protein content in cell lysis or supernatant was labeled with an anti-puromycin antibody. Puromycin intensity in the supernatant and the whole cell lysis was quantified using Image Lab software (Bio-Rad; mean ± SEM, n = 4). **, P < 0.05 (Student’s t test). Representative immunoblot is displayed in Fig. S5. a.u., arbitrary units.](https://cdn.rupress.org/rup/content_public/journal/jcb/218/7/10.1083_jcb.201805002/9/jcb_201805002_fig5.jpeg?Expires=1771571833&Signature=vjpmxSC~9ZoTHCB3ORP4HaDxIfPTlO0gLxLRJ~Z5vLrjoheBmSOJaXJpx4j7jHZY6ql64~uWTz0nas9fplK6bRxZ1F6ROk-jkdLSvyuTwvUauRRsglOnkSRSZGySSNlK-r2z9YCqm3F8HcqDXqWS4k-Fj6bnHxxgUYsVnIgyYq-MOpZVk7jx4e8IwEnfhrPpfHkJoQoFqyqZ9DHaOLEz-V1UBCzHTZFuzY6Pi73rThJe5f5pHPQwGcd9-Rd4S7W2LsLhmmOpj9YaH-JRZf-u9NHtIU3W~UweAu1EWTEQC2GR6b9jdv~VimgAQKBEP2stDsUCKh2tLvhABns4vQMXuA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RAB6 and KIF5B are required for the proper secretion SBP-EGFP-CD59, TNFα-SBP-EGFP, and SBP-EGFP-ColX at the plasma membrane; RAB6 depletion delays total protein secretion. (A) Left: HeLa-TNFα-SBP-EGFP, treated or not with RAB6 siRNAs, were incubated for 30 or 60 min with biotin to allow cargo release. The amount of secreted TNFα was determined on images acquired on cells using the SPI assay. Representative images of cells expressing TNFα-SBP-EGFP (green) and stained with anti-SBP (red) are displayed. Right: Quantification of the amount of TNFα present at the plasma membrane in cells treated as above (mean ± SEM, n = 67–111 cells). **, P < 10−2 (Student’s t test). Scale bars, 5 µm. (B) Left: HeLa-SBP-EGFP-ColX treated or not with RAB6 siRNAs were incubated for 30, 60, or 120 min with biotin to allow cargo release from the ER. The amount of secreted cargo was revealed by Western blotting using an anti-GFP antibody. β-tubulin was used as a loading control and as a nonsecreted protein. Representative immunoblots are displayed. Right: Quantification of the amount of ColX present at the plasma membrane in cells treated as indicated above (mean ± SEM, n = 3). *, P < 0.05 (Student’s t test). (C) HeLa cells expressing SBP-EGFP-CD59, TNFα-SBP-EGFP, and SBP-EGFP-ColX were treated for 3 d with control, RAB6, or KIF5B siRNAs. Cells were incubated for 30 min with biotin to allow cargo release from the ER. Cells were imaged using a time-lapse spinning disk confocal microscope. Vesicle velocity (µm/s) and the number of vesicles per cell were quantified using ImageJ (mean ± SEM, n = 17–35 cells). *, P < 0.05; **, P < 0.005; ***, P < 10−4 (Student’s t test). (D) The SUnSET assay was used to determine the effect of RAB6 depletion on total protein secretion. MEFs prepared from RAB6 loxP/Ko Rosa26CreERT2-TG embryos (described in Bardin et al. [2015]; named after MEF RAB6lox/lox) were treated with ethanol or with 4-OHT for 96 h to induce RAB6 depletion. Cells were then incubated with puromycin and chased in puromycin-free medium for 0, 1, 2, 4, or 5.5 h. Total protein content in cell lysis or supernatant was labeled with an anti-puromycin antibody. Puromycin intensity in the supernatant and the whole cell lysis was quantified using Image Lab software (Bio-Rad; mean ± SEM, n = 4). **, P < 0.05 (Student’s t test). Representative immunoblot is displayed in Fig. S5. a.u., arbitrary units.
