Fig. 3.

Constitutive and accelerated KIM-1 shedding is inhibited by TNF-α-converting enzyme (TACE) inhibitors and short hairpin (sh) RNA-mediated knockdown of TACE. A: confluent monolayers of 769-P renal cell carcinoma (RCC) cells were first pretreated with DMSO (control), GM6001 (1 μM), or TAPI-0 (10 μM) for 30 min following exposure to H₂O (1 μl) or H₂O₂ (1 mM) for 30 min. Shed KIM-1 was detected in media with AKG antibody. Cleaved and total KIM-1 was detected in total cellular lysate with 195 antibody. B and C: experiment in A was repeated with PMA (1 μM) for 1 h and PV (50 μM) for 30 min. A and B: soluble KIM-1 relative to total KIM-1 was quantified by densitometry. Lines between the bands represent any lanes that were rearranged from the original blot. Cleaved KIM-1 was detected using a 15% gel, while total KIM-1 and actin were detected using a 10% gel. D: cell lysates of 786-O RCC cells stably expressing a vector encoding shRNA targeting TACE (shTACE) or the empty vector alone (pSilencer) were analyzed by Western blotting with anti-TACE antibody. A band corresponding to TACE is shown. E: confluent monolayers shTACE and pSilencer cells were preincubated with PMA for 30 min, and KIM-1 shedding was assessed by Western blot analysis. Actin was used as a loading control. The Western blot data are representative of the results obtained from 3 independent experiments. F: 769-P cells were incubated with H₂O (1 μl), H₂O₂ (1 mM), DMSO, or PMA (1 μM) for 30 and 60 min, respectively, before detection of surface TACE expression by flow cytometry using a TACE antibody. Surface TACE expression is displayed in the form of a histogram against the % maximum mean fluorescent intensity and as a graph representative of data from 3 independent experiments. *P < 0.05.