Localization of endogenous BIG1 and BIG2 in COS7 cells by immunofluorescence microscopy during Tfn uptake. Cells were incubated without serum for 3 h and then with Alexa Fluor 568-tagged Tfn (red) at 37°C for 2, 10, 30, or 60 min before fixation and staining with anti-BIG 1 or anti-BIG2 antibodies, followed by Alexa Fluor 488-conjugated anti-rabbit IgG. The experiment was repeated three times. (Scale bar, 16 μm.)

Separation of EEs and REs by Optiprep gradient centrifugation. (A) Cells grown to 70% confluence on 100-mm culture plates were incubated for 3 h without serum and then with Alexa Fluor 488-tagged Tfn at 37°C for 2 or 60 min before rapid chilling, washing, and homogenization. Samples of proteins (10 μg) in postnuclear supernatant before gradient fractionation were separated in 4–12% NuPAGE Bis-Tris gel, followed by immunoblotting and densitometric quantification of BIG1, BIG2, Rab11, Rab4, Exo70, and TfnR. Data are expressed relative to those of the same protein in cells at zero time = 100 and are reported as means ± SD of values from three experiments, with blots from a representative experiment. ∗, P < 0.05. (B) Homogenates of cells prepared as described in A were centrifuged for 5 min at 1,000 × g. Samples (1 mg/ml) of supernatants were applied to 5–20% iodixanol gradients (10 ml), which were centrifuged. Samples of the indicated fractions (collected from the bottom) were analyzed by immunoblotting with antibodies against the indicated proteins. The experiment was repeated three times.

Effects of BIG1 and BIG2 siRNA and controls on protein levels. (A) Cells (1.5 × 10⁵ cells per well) in six-well plates were incubated without or with siRNA as indicated. After 72 h of siRNA treatment, some cells were washed and incubated in complete DMEM for 24 or 48 h. Proteins (35 μg) in samples of cell lysates [sonified in 1 ml of buffer (20 mM Tris·HCl, pH 8.0/1 mM EDTA/1 mM NaN₃ 2 mM MgCl₂/2 mM DTT/250 mM sucrose)] were separated in 4–12% NuPAGE Bis-Tris gel, followed by immunoblotting with antibodies against BIG1 or BIG2. Control lanes: 1, untreated; 2, mock-transfected (vehicle only); 3, nontarget siRNA. siRNA lanes: BIG1 or BIG2 treatment for 24, 48, or 72 h. After siRNA lanes: 24 or 48 h after removal of siRNA following 72-h treatment. (B) As in A, cells were incubated for 72 h with or without the indicated siRNA, and proteins were immunoblotted with antibodies against BIG1, BIG2, ARF, or actin. Lanes: 1, untreated; 2, vehicle only; 3, non-target siRNA; 4, BIG1 siRNA; 5, BIG2 siRNA. Data were similar in more than two other experiments. (C) Data are means ± SD of values from three experiments like that in B. Amounts of the indicated proteins were quantified by densitometry and expressed relative to that of the same protein in untreated normal cells = 100.

Effects of BIG1 and BIG2 siRNA on subcellular distribution of TfnR. (A) Cells (2 × 10⁴ cells per well) in four-well culture slides were incubated for 3 h without serum after 72 h in complete medium with or without the indicated siRNA before confocal immunofluorescence microscopy with antibodies against TfnR and giantin (a cis- and medial-Golgi marker). (Scale bar, 8 μm.) (B) Proteins (10 μg) from postnuclear supernatant of cells from five 100-mm plates incubated for 72 h with or without the indicated siRNAs were separated in 4–12% NuPAGE Bis-Tris gel, followed by immunoblotting with antibodies against BIG1, BIG2, TfnR, and actin. Lanes: 1, mock-transfected; 2, non-target siRNA; 3, BIG1 siRNA; 4, BIG2 siRNA; 5, BIG1 and BIG2 siRNA. (C) Data are means ± SD of values from three experiments like that in B. TfnR was quantified by densitometry of immunoblots and expressed relative to that of the same protein in mock-transfected cells. ∗, P < 0.01 vs. mock.

Effects of BIG1 and BIG2 siRNA on Tfn uptake and release. (A) After incubation for 72 h with vehicle (mock) or the indicated siRNA, cells (1 × 10⁴ cells per well) in 24-well plates were incubated at 37°C overnight in complete DMEM and then without serum for 3 h before addition of Alexa Fluor 488-tagged Tfn. At the indicated time thereafter, cells were washed extensively and lysed. Alexa Fluor 488-tagged Tfn in lysates was quantified by ELISA. Data are means ± SD of values from three experiments. ∗, P < 0.01 vs. mock. (B) Cells incubated with Alexa Fluor 488-tagged Tfn for 60 min as described in A were washed extensively, stripped of surface-bound Tfn, and incubated in DMEM containing unlabeled Tfn. At the indicated time, medium and cells were collected separately, Alexa Fluor 488-tagged Tfn in samples of cell lysate and medium was quantified by ELISA, and the amount in medium was expressed as the percentage of the total in medium plus cell lysate. Data are means ± SD of values from three experiments. ∗, P < 0.01 vs. mock. (C) Cells (2 × 10⁴ cells per well) in four-well culture slides were incubated at 37°C for 72 h without or with siRNA as indicated, for 3 h without serum, and then with Alexa Fluor 568-tagged Tfn for 60 min. After being washed extensively, cells were incubated in DMEM containing unlabeled Tfn at 37°C for the indicated time before microscopy. (Scale bar, 20 μm.)