Supplemental Material

This article contains the following supporting material:

• Figure 1 - RINT-1N is almost exclusively localized in the cytosol and binds ZW10 but not syntaxin 18 or BNIP1. (A) 293T cells grown in a 60-mm dish were transfected with the plasmid for FLAG-tagged full-length RINT-1 or RINT-1N. At 24 h after transfection, the cells were collected, suspended in 1 ml homogenization buffer (20 mM Hepes-KOH, pH 7.2, 150 mM KCl, and 2 mM EDTA) and then homogenized by passing through a 26-gauge needle 20 times. The homogenate was centrifuged at 1,000 x g for 5 min, and then the supernatant was centrifuged at 100,000 x g for 30 min to separate the cytosol and membrane fraction. The same amount (each 10 μg) of the post-nuclear supernatant (PNS) (lanes 1 and 4), cytosol (lanes 2 and 5), and total membranes (TM) (lanes 3 and 6) were analyzed by immunoblotting with the indicated Abs. (B) 293T cells grown in a 35-mm dish were transfected with the plasmid for FLAG-tagged full-length RINT-1 or RINT-1N. At 24 h after transfection, cells lysates were prepared and FLAG-tagged proteins were immunoprecipitated with an anti-FLAG Ab. The immunoprecipitaed proteins were analyzed by immunoblotting with the indicated Abs (lanes 3 and 4). Input (4 % of total) was also analyzed (lanes 1 and 2).

• Figure 2 - The transport of VSVG-GFP is blocked at the ERGIC in RINT-1N-overexpressing cells. The plasmid for VSVG-GFP was co-transfected with the plasmid for FLAG-RINT-1N into HeLa cells. The cells were incubated at 40°C for 24 h, and then 32°C for 1 h. The cells were fixed and double-stained with an anti-Sec31p, β-COP, or ERGIC-53 Ab followed by a Texas Red-conjugated secondary Ab, and an anti-FLAG Ab followed by an Alexa-350-conjugated secondary Ab. The latter staining allowed us to identify FLAG-RINT-1N expressing cells. Enlarged images from the areas indicated by white boxes are shown below each panel. Bar, 5 μm.

• Figure 3 - Dispersion of Sec31p and ERGIC-53 in RINT-1-depleted cells. Samples were prepared as described in the legend to Figure 4 and immunofluorescence images were obtained by scanning at lower PMT voltage. Bars, 5 μm.