Figure 6.
Figure 6. Covalent cross-linking of SNAP-uLCa disrupts CHC17-dependent pathways of transferrin uptake and recycling. (A) HeLa-SNAP-uLCa clone 3.3 cells were treated with SNAP-tag homodimerizer (BG-GLA-BG, 10 µM) or mock treated (0 µM) with solvent (DMSO) equivalent to the amount used for the BG-GLA-BG treatment. Cell lysates were immunoblotted for dimerized SNAP-uLCa (114** kD), monomeric SNAP-uLCa (57* kD), and endogenous uLCa (32 kD) using antibodies to the SNAP-tag or LCa (mAb X16), as indicated (left). β-actin was detected as a loading control. Protein mass based on migration relative to marker proteins is shown on the right. (B) Clone 3.3 cells were treated (2 h) with 5 µM BG-GLA-BG or equivalent DMSO (mock). Internalization of prebound (4°C) Alexa Fluor 488–transferrin (Tf) was visualized after 10 min or 1 h at 37°C after IF labeling with antibodies to the SNAP-tag or CHC17. Red-blue colocalization in merge is pink. Three-color colocalization in merge is white. Bars, 10 µm. (C) Clone 3.3 cells internalized prebound Alexa Fluor 488–transferrin for 1 h and were labeled by IF with antibodies to the SNAP-tag and EEA1. Red-green colocalization in merge is yellow. Three-color colocalization in merge is white. Bars, 10 µm. (B and C, insets) Boxes are magnified threefold. Bars, 1 µm. Images are 2D. (D) Clone 3.3 cells were treated with BG-GLA-BG or DMSO (mock), as in B, labeled with NHS-S-S-biotin, and then kept on ice (0* and 0 min) or incubated at 37°C for 5, 10, or 15 min. After reduction (stripping) of all samples except 0*, cells were lysed and analyzed by immunoblotting for total TfR and GAPDH (lysate), or biotinylated proteins were SA bound and immunoblotted to detect internalized TfR or control GAPDH. Protein mass based on migration relative to marker proteins is shown on the right. (E) TfR uptake (signal at internalization time point minus signal at time 0) was quantified by densitometry and plotted relative to total biotinylated TfR (0* minus signal at time 0) for four independent experiments, performed as in D for clone 3.3 cells treated with BG-GLA-BG (shaded squares) or DMSO (mock; open circles). Internalized TfR is shown in arbitrary units. Data represent the mean ± SEM of three separate experiments.

Covalent cross-linking of SNAP-uLCa disrupts CHC17-dependent pathways of transferrin uptake and recycling. (A) HeLa-SNAP-uLCa clone 3.3 cells were treated with SNAP-tag homodimerizer (BG-GLA-BG, 10 µM) or mock treated (0 µM) with solvent (DMSO) equivalent to the amount used for the BG-GLA-BG treatment. Cell lysates were immunoblotted for dimerized SNAP-uLCa (114** kD), monomeric SNAP-uLCa (57* kD), and endogenous uLCa (32 kD) using antibodies to the SNAP-tag or LCa (mAb X16), as indicated (left). β-actin was detected as a loading control. Protein mass based on migration relative to marker proteins is shown on the right. (B) Clone 3.3 cells were treated (2 h) with 5 µM BG-GLA-BG or equivalent DMSO (mock). Internalization of prebound (4°C) Alexa Fluor 488–transferrin (Tf) was visualized after 10 min or 1 h at 37°C after IF labeling with antibodies to the SNAP-tag or CHC17. Red-blue colocalization in merge is pink. Three-color colocalization in merge is white. Bars, 10 µm. (C) Clone 3.3 cells internalized prebound Alexa Fluor 488–transferrin for 1 h and were labeled by IF with antibodies to the SNAP-tag and EEA1. Red-green colocalization in merge is yellow. Three-color colocalization in merge is white. Bars, 10 µm. (B and C, insets) Boxes are magnified threefold. Bars, 1 µm. Images are 2D. (D) Clone 3.3 cells were treated with BG-GLA-BG or DMSO (mock), as in B, labeled with NHS-S-S-biotin, and then kept on ice (0* and 0 min) or incubated at 37°C for 5, 10, or 15 min. After reduction (stripping) of all samples except 0*, cells were lysed and analyzed by immunoblotting for total TfR and GAPDH (lysate), or biotinylated proteins were SA bound and immunoblotted to detect internalized TfR or control GAPDH. Protein mass based on migration relative to marker proteins is shown on the right. (E) TfR uptake (signal at internalization time point minus signal at time 0) was quantified by densitometry and plotted relative to total biotinylated TfR (0* minus signal at time 0) for four independent experiments, performed as in D for clone 3.3 cells treated with BG-GLA-BG (shaded squares) or DMSO (mock; open circles). Internalized TfR is shown in arbitrary units. Data represent the mean ± SEM of three separate experiments.

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