Clone d69c12

For detection of laminin-α4, conditioned media was prepared by concentrating serum-free supernatant obtained from cultured cancer cells incubated for 24hr using 100k cutoff Amicon Ultra (Millipore) centrifuge tubes. For detection of p27, p21, and β-tubulin, cell lysates were collected from cells that had been cultured overnight in Ultra-Low Attachment Surface 6-well plates (Corning). Protein from conditioned media or cell lysates were separated using SDS–polyacrylamide gel electrophoresis, transferred to Immobilon-P Transfer Membrane (Millipore), and probed with antibodies against human laminin-α4 (1:200; clone 6C3, sc-130541, Santa Cruz Biotechnology) or p27 (1:1,000; clone D69C12, Cell Signaling Technology), p21 (1:1,000; clone 12D1, Cell Signaling Technology), or β-tubulin (1:5,000; clone 9F3, Cell Signaling Technology). Primary antibody was chemiluminescently detected using horseradish peroxidase–conjugated secondary antibody (1:10,000), ECL 2 Western Blotting Substrate (Pierce) and the SRX-101A (Konica Minolta) developer. Quantification of western blots was performed using ImageJ (NIH) software. p27 signal intensity was normalized to β-tubulin signal intensity.