Characterization of 1G8 antibodies. (a) Whole cell lysates from the indicated breast epithelial cell lines were resolved by 8% SDS-PAGE and blotted with commercially obtained anti-MUC4 antibody 1G8 (1G8c). MCF7-Rep5 is a human breast cancer line stably expressing inducible rat MUC4 (24). (b) Cell lysates were resolved by 6% to 12% gradient SDS-PAGE and immunoblotted with supernatant from cultured 1G8 hybridoma (1G8s) or with anti-tubulin. The MATB-III rat mammary adenocarcinoma cell line was used as a positive control for MUC4 expression (12, 44, 45). (c) Wild-type JIMT-1 or JIMT-1 cells stably transduced with MUC4 shRNA were blotted with 1G8s and actin. (d) HEK293T cells were transiently co-transfected with pLenti vector control or pLenti-hMUC4/Y vector, together with pSuper vector control (v) or MUC4 shRNA (M4) or scrambled (scr) oligonucleotides in pSuper. Lysates were blotted with 1G8s and actin.