L1 proteolysis in breast cancer cell lines. (A) Western blot analysis of L1CAM expression in MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells. 30 μg extracts from each cell line were probed with NCAM-L1 (C-20) for L1. A fragment around 30 kDa after cleavage is shown (arrow) in MDA-MB-435 and MDA-MB-231 cell extracts. (B) Activation of L1 shedding by PMA. Confluent cells after one day in culture were incubated at 37°C for 1 hr with or without 100 ng/mL PMA. Cell culture supernatants were then collected and TCA precipitated, and cell pellets were lysed respectively. Monoclonal antibody UJ127 was used to probe L1 in all samples. NC, plain QT6 cells as negative control. PC1 and PC2, glioma cell lines U-87 MG and T98G as positive controls.