Journal: American Journal of Physiology - Cell Physiology

Article Title: Hemoglobin inhibits albumin uptake by proximal tubule cells: implications for sickle cell disease

doi: 10.1152/ajpcell.00021.2017

Figure Lengend Snippet: Hemoglobin acutely inhibits receptor-mediated endocytosis in proximal tubule cells. A: HK-2 cells were incubated with 50 µM OxyHb for 4 h or 3 days as indicated, then solubilized and blotted to detect heme oxygenase 1 (HO-1) and β-actin (as a loading control) Quantitation of HO-1 expression (avg ± range) relative to control and normalized to β-actin in two independent experiments is shown next to the blot. B: filter-grown OK cells were preincubated at 37°C with apically added OxyHb (10–50 µM) for 18 h to 5 days as indicated prior to extensive washout and subsequent addition of Alexa Fluor 647-albumin for 1 h at 37°C in the absence of OxyHb. As a positive control, OxyHb (10 µM) was included during the albumin uptake period; untreated cells incubated with Alexa Fluor 647-albumin were used as a negative control. Fluorescent cell-associated albumin was quantified by spectrofluorimetry. Data (means ± SD) from three independent experiments each performed in triplicate are shown.

Article Snippet: HK-2 ( 5 × 10 5 ) cells were cultured as above in the presence of 50 µM OxyHb for the indicated periods, then washed, solubilized, and lysates blotted using rabbit polyclonal anti HO-1 antibody (1:1,500; Abcam ab137749) and mouse monoclonal β-actin antibody (1:5,000; Sigma A1978).

Techniques: Incubation, Quantitation Assay, Expressing, Positive Control, Negative Control