Horseradish peroxidase linked secondary antibody against rabbit or mouse

Immunoblot was performed as described previously (Jin et al., 2013 (link)). Briefly, proteins were loaded and separated on SDS NuPAGE Novex 4-12% gels (Invitrogen, Carlsbad, CA). They were then transferred to the polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was blocked in a blocking buffer (5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween 20) for 1 h, washed and incubated in the blocking buffer containing a primary rabbit or mouse antibody overnight at 4°C. The membrane was washed and incubated for 1 h in a horseradish peroxidase-linked secondary antibody against rabbit or mouse (Jackson Immunoresearch Laboratory, West Grove, PA). Immunoblots were visualized by the enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Piscataway, NJ) with a MagicMark XP Western protein standard (Invitrogen) served for protein size determination. Optical density of immunoblots was measured using NIH gel analysis software. Values of optical density were normalized to actin or tubulin.

Western blots were performed as described previously (Jin et al., 2013 (link)). Briefly, we separated proteins on SDS NuPAGE Novex 4–12% gels (Invitrogen, Carlsbad, CA) and then transferred proteins from gels to polyvinylidene fluoride membranes (Millipore, Bedford, MA). After membranes were blocked and washed, membranes were incubated in a buffer containing a primary rabbit or mouse antibody overnight at 4°C. Membranes were then washed and incubated in a horseradish peroxidase-linked secondary antibody against rabbit or mouse (Jackson Immunoresearch Laboratory, West Grove, PA). We visualized Immunoblots by an enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Piscataway, NJ). Optical density of immunoblots was measured using NIH ImageJ analysis software (RRID: nif-0000-30467). Values of optical density of pY416, Src and Fyn were normalized to a loading control (actin or tubulin) and were reported separately. The pY416 values were not normalized to either Src or Fyn.