HEK293T cells (Clontech, tested negative for mycoplasma contamination by
MycoProbe Mycoplasma Detection Kit (R&D Systems)) were transfected with
pcDNA3–3xFLAG-Ret and/or pAP-VSV-G-Gas1/Gas1-domain-truncated mutants by
lipofectamine2000 (Invitrogen) for 24 h according to manufacturer’s
guidelines. Cells were lysed in lysis buffer (50mM Tris pH 8.0, 150mM NaCl, 0.5%
Digitonin (Sigma), and protease inhibitors) for 1 h at RT. Cell lysates were
cleared by centrifugation, and subjected to immunoprecipition by either
anti-FLAG M2 Affinity Gel (Sigma) or anti-VSV-G Agarose Conjugate (Sigma) for 2
h at 4 °C. Affinity matrixes were washed and eluted in 2X SDS-PAGE sample
buffer for Western blotting using anti-FLAG (Sigma) and anti-VSV-G antibodies
(Invitrogen). Western results were obtained by Odyssey CLx Near-Infrared
Fluorescence Imaging System (Li-Cor) using Image Studio 5 software.
MycoProbe Mycoplasma Detection Kit (R&D Systems)) were transfected with
pcDNA3–3xFLAG-Ret and/or pAP-VSV-G-Gas1/Gas1-domain-truncated mutants by
lipofectamine2000 (Invitrogen) for 24 h according to manufacturer’s
guidelines. Cells were lysed in lysis buffer (50mM Tris pH 8.0, 150mM NaCl, 0.5%
Digitonin (Sigma), and protease inhibitors) for 1 h at RT. Cell lysates were
cleared by centrifugation, and subjected to immunoprecipition by either
anti-FLAG M2 Affinity Gel (Sigma) or anti-VSV-G Agarose Conjugate (Sigma) for 2
h at 4 °C. Affinity matrixes were washed and eluted in 2X SDS-PAGE sample
buffer for Western blotting using anti-FLAG (Sigma) and anti-VSV-G antibodies
(Invitrogen). Western results were obtained by Odyssey CLx Near-Infrared
Fluorescence Imaging System (Li-Cor) using Image Studio 5 software.