Ab197772

Immunoprecipitation was performed according to our previous publications 19 (link). Briefly, protein from HBE cells was extracted with weak RIPA lysis buffer. An appropriate dilution of anti-ITGB4 antibody (Ab197772, Abcam, USA) or anti-EGFR antibody (sc-373746, Santa Cruz, USA) were added to the centrifuge tube which conjugate with suspension of Protein G. Mix the antibody-bead mixture at 4°C for 4 h using tube rotator. Then, 50μg of cell lysates were added into the mixtures and the lysate-bead/antibody conjugate mixtures were incubated overnight at 4°C. Mixtures were further resuspended in 5xSDS loading buffer. Samples were boiled for 5 minutes and analyzed by Western blot. Immunocomplexes were stained with anti-ITGB4 (Ab197772, Abcam, USA) as well as EGFR (sc-373746, Santa Cruz, USA).

In brief, protein was extracted with radioimmunoprecipitation assay (RIPA) buffer: 50 mmol/L Tris‐HCl, 1% NP40, 0.5% sodium deoxycholate, 150 mmol/L NaCl, 0.1% SDS, 5 mmol/L EDTA, pH = 7.4.38 The immune complexes were allowed to bind to Protein A‐Sepharose beads for 3 hours at 4°C, washed (15 minutes, 4°C) with RIPA buffer and resuspended in loading buffer. Immunoprecipitates were then subjected to 10% SDS‐PAGE and immunoblotted on a polyvinylidene difluoride membrane. Immunocomplexes were stained with anti‐ITGB4 (Ab197772, Abcam) as well as EGFR (Ab52894, Abcam) by use of ECL (NCI4106, Thermo Pierce).

Immunofluorescence (IF) was detected and analyzed according to our previous publication (27 (link)). The following primary antibodies were used: ITGB4 (ab197772, Abcam, USA), RSV-F (ab24011, Abcam, USA).