Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling Compartment -- Strick and Elferink 16 (12): 5699 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling Compartment
Mol. Biol. Cell Strick and Elferink 16: 5699

Supplemental Material

This article contains the following supporting material:

Figure S1 - RT-PCR analysis of endogenous REP15 and Rab15 expression in HeLa cells.
A
, Endogenous mRNA levels of REP15 and Rab15 were measured by RT-PCR, using specific primers (50 pmol) with 5 ug of Total RNA isolated from HeLa cells. β-actin levels were determined as an internal control, using actin specific primers. B, Endosomal membrane fractions prepared from untransfected Hela cells (Con) and cells stably expressing cMyc-tagged REP15 (REP15) were analyzed by Western analysis for REP15, cMycREP15 (cMyc) and TfR.
Figure S2 - An amino-terminal cMyc epitope does not interfere with REP15 and Rab15-GTP colocalization.
ƒn HeLa cells were cotransfected with cMycREP15 and Rab15 wt or Q67L (GTP-bound) and examined using confocal microscopy. Scale, 10 μm.
Figure S3 - Comparable levels of B-Tfn are internalized into the ERC of control and REP15 over expressing HeLa cells.
The ERC of control and REP15 over expressing HeLa cells were loaded with B-Tfn using a combination of pulse chase and acid wash conditions as described in the Materials and Methods. The cells were lysed and the amount of internalized B-Tfn in 1 ug of the cell lysate was determined using the B-Tfn ELISA. Values represent the mean percentage of B-Tfn loaded into the ERC ± S.E.
Figure S4 - Internalized TfR is retained in cells transiently over expressing REP15.
A
, The level of intracellular TfR retained in the ERC of HeLa cells over expressing REP15 (REP15) and mock-transfected cells (Con) was quantified using surface biotinylation assays. The percentage (%) of intracellular TfR retained in the ERC was calculated as 100(D_t)/D₀ where D_t is the quantity of biotinylated TfR remaining following cleavage at any given time and D₀ is the total amount of biotinylated TfR loaded into the ERC at chase time 0 min. All values represent the mean ± S.E. of four independent assays. Significant Differences (* p<0.01, ANOVA) were observed between experimental and control conditions. B, Representative Western blots are shown.