Figure S1.
GRAF1b/2 colocalize with Rab8a/b and Rab10 in HeLa cells. (A and B) Confocal images of transfected live cells showing overlapping (RFP–Rab8a/b) or contiguous (RFP-Rab10) colocalization with GRAF1b/2–GFP on tubules. (B) Snapshots were taken every 5 s. Regions with dynamic tubules are shown at relevant time points. Rab8a/b and GRAF1b/2 appeared simultaneously (white arrows), but Rab10 (red arrows/arrowheads) preceded GRAF1b (cyan arrows/arrowheads). Scale bars: 5 µm. (C) Confocal images of cells transfected with GRAF1b-GFP and stained with α-Rab8 showing colocalization. (D) Immunoprecipitation (IP) of GRAF proteins from an equal amount of rat brain lysate using a purified antibody against GRAF1 (α-GRAF1), a purified antibody against GRAF2 (α-GRAF2), or unpurified serum detecting both GRAF1 and GRAF2 (α-GRAF1/2). Pre-immune serum was used as negative control. Top and bottom membranes are replicates. GRAF2 migrates at a higher molecular weight than all GRAF1 isoforms. (E) GRAF proteins were immunoprecipitated from an equal protein amount of HeLa, NIH 3T3, and 293T cell lysates using α-GRAF2 (top) or α-GRAF1 (middle). Actin was used as loading control on the corresponding lysates (bottom). IP with α-GRAF2 showed GRAF2 expression in the three cell lines. IP with α-GRAF1 showed GRAF1 expression in 293T cells. On this membrane, the faint band seen in HeLa and NIH 3T3 samples (*) corresponded to traces of GRAF2 protein, as it was at a higher molecular weight than the GRAF1 band and its intensity was increased after stripping and reprobing of the membrane with α-GRAF2. This suggests that in lysates containing low amounts of GRAF1, α-GRAF1 can cross-react with GRAF2. (F) Equal amounts of a HeLa cell lysate were immunoprecipitated with an α-GRAF1 antibody described in earlier studies (Ra83), pre-immune serum, or α-GRAF1/2 serum. Proteins bound to beads were separated by electrophoresis and stained with Coomassie. The region around the mol wt for GRAFs was cut and analyzed by LC-MS/MS. Lanes 2 and 4 contained GRAF2 (Uniprot accession no. A1A4S6), but no GRAF1 peptides were identified. (G) IPof endogenous GRAF2 from an equal amount of shRNA-transfected HeLa cell lysates. Beads and lysates were analyzed in parallel, showing knockdown of GRAF2 in shGRAF2a- and shGRAF2b-transfected cells. Actin was used as loading control. (H) Confocal stacks of HeLa cells expressing shControl or shGRAF2a and transfected with RFP-Rab8a or RFP-Rab10. (I) Confocal images of transfected live cells. GRAF1b-GFP was diffuse when expressed with RFP-Rab8a T22N but colocalized with RFP–Rab8a Q67L. (A, C, H, and I) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

GRAF1b/2 colocalize with Rab8a/b and Rab10 in HeLa cells. (A and B) Confocal images of transfected live cells showing overlapping (RFP–Rab8a/b) or contiguous (RFP-Rab10) colocalization with GRAF1b/2–GFP on tubules. (B) Snapshots were taken every 5 s. Regions with dynamic tubules are shown at relevant time points. Rab8a/b and GRAF1b/2 appeared simultaneously (white arrows), but Rab10 (red arrows/arrowheads) preceded GRAF1b (cyan arrows/arrowheads). Scale bars: 5 µm. (C) Confocal images of cells transfected with GRAF1b-GFP and stained with α-Rab8 showing colocalization. (D) Immunoprecipitation (IP) of GRAF proteins from an equal amount of rat brain lysate using a purified antibody against GRAF1 (α-GRAF1), a purified antibody against GRAF2 (α-GRAF2), or unpurified serum detecting both GRAF1 and GRAF2 (α-GRAF1/2). Pre-immune serum was used as negative control. Top and bottom membranes are replicates. GRAF2 migrates at a higher molecular weight than all GRAF1 isoforms. (E) GRAF proteins were immunoprecipitated from an equal protein amount of HeLa, NIH 3T3, and 293T cell lysates using α-GRAF2 (top) or α-GRAF1 (middle). Actin was used as loading control on the corresponding lysates (bottom). IP with α-GRAF2 showed GRAF2 expression in the three cell lines. IP with α-GRAF1 showed GRAF1 expression in 293T cells. On this membrane, the faint band seen in HeLa and NIH 3T3 samples (*) corresponded to traces of GRAF2 protein, as it was at a higher molecular weight than the GRAF1 band and its intensity was increased after stripping and reprobing of the membrane with α-GRAF2. This suggests that in lysates containing low amounts of GRAF1, α-GRAF1 can cross-react with GRAF2. (F) Equal amounts of a HeLa cell lysate were immunoprecipitated with an α-GRAF1 antibody described in earlier studies (Ra83), pre-immune serum, or α-GRAF1/2 serum. Proteins bound to beads were separated by electrophoresis and stained with Coomassie. The region around the mol wt for GRAFs was cut and analyzed by LC-MS/MS. Lanes 2 and 4 contained GRAF2 (Uniprot accession no. A1A4S6), but no GRAF1 peptides were identified. (G) IPof endogenous GRAF2 from an equal amount of shRNA-transfected HeLa cell lysates. Beads and lysates were analyzed in parallel, showing knockdown of GRAF2 in shGRAF2a- and shGRAF2b-transfected cells. Actin was used as loading control. (H) Confocal stacks of HeLa cells expressing shControl or shGRAF2a and transfected with RFP-Rab8a or RFP-Rab10. (I) Confocal images of transfected live cells. GRAF1b-GFP was diffuse when expressed with RFP-Rab8a T22N but colocalized with RFP–Rab8a Q67L. (A, C, H, and I) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

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