Ist1 is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 (IST1 knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. GAPDH immunoblotting serves as a loading control. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in d. (e) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were labeled for TfnR. Increased TfnR tubulation is seen in the IST1-depleted cells (see arrowheads in inset magnified region of the boxed area). The exposure settings in the IST1 knockdown image have been increased to compensate for the reduced TfnR levels in these cells. (f–h) Confocal micrographs of HeLa cells subjected to mock transfection (f) or transfected with IST1 siRNA1 (g and h) and then labeled with the markers shown. Confocal micrograph gain settings were identical in f and g, but in h, which shows higher magnification images of the dashed areas indicated in g, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Insets are magnified areas of the boxed regions. (i–k) Uptake of Alexa Fluor Tfn 647 was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with spastin siRNA1 (as a positive control), and IST1 siRNA1 (i) or IST1 siRNA3 (j). Mean Tfn uptake at the 20-min time point is shown (n = 3 experiments plotted for each histogram). A representative time course experiment is shown in k. Bars, 10 µm. Error bars show SEMs.