Figure 5
From: A mass spectrometry-based approach for the identification of Kpnβ1 binding partners in cancer cells

Validation of Kpnβ1 binding partners identified by IP-MS. (A) Expression of known Kpnβ1 interactors, Kpnα2 and Ran, and CRM1, was analysed in hTERT-RPE-1, HeLa, WHCO5 and KYSE30 whole cell lysates (30 µg) by Western blot analysis. GAPDH was used to control for protein loading. (B) Co-immunoprecipitation assays were performed and Kpnα2, Ran and CRM1 proteins detected by Western blot analysis. GAPDH was used as a negative control, as it is not expected to bind Kpnβ1. CRM1, Kpnα2 and Ran proteins were not pulled down in lysates incubated with a non-specific IgG isotope control and protein A agarose beads. 10 µg HeLa protein lysate was included as a positive control. (C) Western blot analysis showing expression of novel Kpnβ1 interactors, CCAR1 and FUBP1 in hTERT-RPE-1, HeLa, WHCO5 and KYSE30 whole cell lysates. GAPDH was used to control for protein loading. (D) Co-immunoprecipitation assays showing successful pull-down of CCAR1 and FUBP1 with Kpnβ1. GAPDH was used as a negative control. CCAR1 and FUBP1 were not pulled down in lysates incubated with a non-specific IgG isotope control and protein A agarose beads. Experiments were performed two times.