2.5. Co‐immunoprecipitation studies

Suspended Caco‐2 cells were treated with vehicle control DMSO and/or 10 nM Zn27 in dishes precoated with 1% heat‐inactivated BSA at 37°C in 8% CO₂. After a 1 h incubation, cells were lysed in a nondenaturing lysis buffer (Pierce IP Lysis Buffer, Cat. No. 87788, Thermo Fisher Scientific Inc) and a 1–2 mg of protein was used for each immunoprecipitation. For immunoprecipitation of FAK, the total protein was incubated with mouse anti‐FAK antibody, at 4°C overnight. Normal mouse IgG was used as a control antibody for the co‐immunoprecipitation. Protein G plus/protein A agarose suspension beads were added to the lysate containing anti‐FAK and incubated for an extra 2 h at 4°C. Then, the beads were washed, and protein was eluted in 6X Laemmli buffer (Alfa Aesar, J61337‐AD, Thermo Fisher Scientific). The eluted protein samples were resolved by 10% SDS‐PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the anti‐FAK antibody for IP. The immunoprecipitated blots were then probed with anti‐PTP1B to assess co‐precipitating PTP1B associated with FAK. The secondary antibody anti‐mouse 800 was used for detection of FAK and the detection of PTP1B, anti‐secondary Ab mouse monoclonal secondary antibody to Rabbit IgG light chain (HRP) was used. The total lysates loaded on gel and western blotted served as input control for the Co‐IP. The images were acquired by LICOR imaging for FAK, whereas the chemiluminescence detection system and Biorad gel doc were used for PTP1B. Densitometric quantitation was performed with LICOR imaging (LI‐COR Biosciences).