A, cells expressing 315-HARE, 190-HARE, or EV alone were cultured in T-25 flasks until confluent, washed, scraped, and lysed. Cell lysates (100ug of protein) were immunoprecipitated (IP) with anti-Tyr(P) mAb and the immune complexes were subjected to 5% SDS-PAGE and Western blot (WB) analysis using anti-V5 pAb. Cells expressing 315-HARE (B) or 190-HARE (C) cDNA were grown until confluent, washed, incubated for 1 h in medium without serum, and then incubated with (HA) or without (Ctr) 5ug/ml HA for 20 min. The cells were lysed and 200 ug of total lysate protein was immunoprecipitated with anti-HARE mAb-30. Immune complexes were collected and subjected to 5% SDS-PAGE and Western analysis using anti- Tyr(P) mAb. The same blots in B and C were reprobed with anti-V5 Ab (D and E, respectively). Immunopurification using non-immune mouse IgG served as another control in B–E. The solid and open arrows indicate the positions of the 315-HARE and 190-HARE, respectively.

All lanes : Anti-V5 tag antibody (ab95038) at 0.4 µg/ml

Lane 1 : E. coli whole cell lysate expressing a multi-tag fusion protein at 0.2 µg
Lane 2 : E. coli whole cell lysate expressing a multi-tag fusion protein at 0.1 µg
Lane 3 : E. coli whole cell lysate expressing a multi-tag fusion protein at 0.05 µg

Developed using the ECL technique.

Exposure time: 30 seconds

Immunohistochemical analysis of murine mammary tumour tissue, expressing vector (left) or V5-tagged Cadm1 (right), staining V5 tag with ab95038.