Sc 46655

A total of 1 × 10⁵ MRC-5 cells cultured on 12-well chamber slides were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The slides were then blocked with 10% goat serum at room temperature for 10 min. Subsequently, the samples were incubated with primary antibodies, including rabbit anti-Thy-1 (ab225, Abcam, US) and mouse anti- integrin β3 (sc-46655, Santa Cruz, USA), respectively, at 4 °C overnight, followed by incubation with Alexa Flour 488 or Cy3 secondary antibodies (Beyotime, China) for 1 h at room temperature. The slides were counterstained with DAPI and examined by fluorescence microscopy (Leica, Heidelberg, Germany). Primary antibodies included rabbit anti-Thy-1 from CST and mouse anti-integrin β3 from Santa Cruz.

Co-immunoprecipitation was performed according to a previous report.¹⁶ Briefly, proteins were extracted with cell lysis buffer (KGP701~KGP701–100, KeyGEN BioTECH, China) with 1% PMSF (KGP610, KeyGEN BioTECH, China). A total of 500 μg of protein extract was then incubated with 2 μg of appropriate immunoprecipitation (IP) antibodies, i.e., rabbit anti-Thy-1 (ab225, Abcam, US) or mouse anti- integrin β3 (sc-46655, Santa Cruz, USA) at 4 °C overnight with shaking. Then, 20 μl of protein A/G plus agarose (Thermo) was added, followed by incubation at 4 °C for 3 h with shaking. The pellets were washed five times with cell lysis buffer for 2 min and resuspended in 40 μl of 2 × electrophoresis loading buffer. After boiling for 10 min, 20 μl samples were used for SDS-PAGE and assessed by Western blot as described above.