Imagequant las 4000 micro instrument

The extracted protein was mixed with loading buffer and separated by electrophoresis in 10% SDS-polyacrylamide gels. The separated proteins were then transferred onto PVDF membranes (#IPVH00010, Merck Millipore, Billerica, MA, USA) and blocked with Tris-Tween-20 (TBST) buffer containing 5% non-fat milk for 1 h at 37 °C. The membranes were incubated overnight at 4 °C with the appropriate antibody. After being washed with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 2 h at 37 °C. All the primary antibodies and secondary antibodies were listed in Tables S1 and S2. The bands were then displayed with Immobilon Western Chemiluminescent HRP Substrate (#WBKLS0100, EMD Millipore Corporation, Burlington, MA, USA) and captured on an ImageQuant LAS 4000 Micro Instrument (GE Healthcare Bio-Science AB, Uppsala, Sweden). The mean grey value of the protein samples was quantified using Image J. In the phos-tag assay, MH7A cells were transfected with indicated plasmids or treated with paroxetine. 48 h post-transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Phosphorylation of SAV1 proteins was detected using gels containing phos-tag Acrylamide AAL-107 (#304-93521, FUJIFILM Wako Pure Chemical Corporation).

The extracted protein was mixed with loading buffer and separated by electrophoresis in 10% SDS-polyacrylamide gels. The separated proteins were then transferred onto PVDF membranes (#IPVH00010, Merck Millipore, Billerica, MA, USA) and blocked with Tris-Tween-20 (TBST) buffer containing 5% non-fat milk for 1 h at 37 °C. The membranes were incubated overnight at 4 °C with the appropriate antibody. After being washed with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 2 h at 37 °C. All the primary antibodies and secondary antibodies were listed in Tables S1 and S2. The bands were then displayed with Immobilon Western Chemiluminescent HRP Substrate (#WBKLS0100, EMD Millipore Corporation, Burlington, MA, USA) and captured on an ImageQuant LAS 4000 Micro Instrument (GE Healthcare Bio-Science AB, Uppsala, Sweden). The mean grey value of the protein samples was quantified using Image J. In the phos-tag assay, MH7A cells were transfected with indicated plasmids or treated with paroxetine. 48 h post-transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Phosphorylation of SAV1 proteins was detected using gels containing phos-tag Acrylamide AAL-107 (#304-93521, FUJIFILM Wako Pure Chemical Corporation).