Volume 8 issue 4 April 2007
Role of an acidic cluster/dileucine motif in cation-independent mannose 6-phosphate
receptor traffic
Lori L. Tortorella, Florencia B. Schapiro and Frederick R. Maxfield

Figure S1: Little Alexa488 quenching antibody is taken up by fluid phase endocytosis. MPR wild-type cells were incubated without (A, B, C) or with (D, E, F) 5 m g/ml Alexa 488-anti-bCI-MPR monoclonal antibody for 8 minutes at 37ºC (A and D). Cells were washed with PBS and then chased in the presence of 25 µg/ml anti-Alexa488 polyclonal antibody for 30 minutes. The cells were washed, fixed with PFA, permeabilized with saponin, and then stained with goat-anti-rabbit IgG-Alexa 546 for 30 minutes to detect the Alexa 488 antibody (B and E). Images were collected on a wide field fluorescence microscope, and representative images are shown for each data set. Only the cells pre-incubated with Alexa 488-anti-bCI-MPR monoclonal antibody take up significant amounts of the quenching antibody (compare panel B to E). These data indicate that the amount of quenching due to fluid phase endocytosis of the quenching antibody would be minimal.

Figure 1 (jpg)

Figure S2: AP-1 does not colocalize to Tf containing tubules or vesicles. The gamma-adaptin-GFP expression plasmid was created using a standard PCR based cloning strategy. First, the full-length gamma adaptin cDNA was cloned from a mouse NIH3T3 cDNA library by reverse transcription (RT)-PCR. NIH3T3 total RNA was isolated using a Qiagen RNAeasy kit and used for first strand cDNA synthesis by SuperscriptII reverse transcriptase (Invitrogen). The gamma adaptin cDNA was amplified from the cDNA pool and then subcloned into the KpnI and BamHI sites of pcDNA3.1+ (Invitrogen). The PCR amplified gamma adaptin cDNA contained a Kozak sequence prior to the initiating methionine and lacked a stop codon. The eGFP cDNA was amplified by PCR using peGFP (Clontech) as a template and then subcloned in-frame to the C-terminal end of gamma adaptin using the BamHI and EcoRI sites in pcDNA3.1+. A 12 nucleotide spacer was included between the gamma adaptin and eGFP cDNA's.

MPR wild-type cells were transfected with 1 µg of gamma adaptin-GFP cDNA per well of a 6 well dish using Lipofectamine 2000 as recommended by the manufacturer. The cells were plated onto coverslip bottom dishes, and 48 hours after transfection were labeled at 37ºC with 5 µg/ml Alexa 546-Tf. Cells were maintained in Alexa 546-Tf during the live cell image acquisition. Shown are representative single confocal planes which show that there is little overlap between gamma adaptin-GFP (green) and transferrin (red) in these cells. Bar, 2.5 µm

Figure 2 (jpg)