Ecl plus immunoblotting chemiluminescence system

Mouse brain tissue and cells were lysed in NP-40 lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40), and protein concentration in the cell lysate was quantified using a Bradford protein assay (Bio-Rad). Protein samples were incubated at 100 °C for 5 min and electrophoresed on 10% SDS-polyacrylamide gels. Proteins were transferred onto PVDF membranes (GE Healthcare Life sciences). Membranes were blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Membranes were then incubated at 4 °C with a primary antibody mouse anti-Eps8 (BD Transduction) and mouse anti-GAPDH (Millipore) diluted in 5% nonfat milk overnight (1:500 and 1:10,000, respectively) then washed several times with TBS-T. After 1 h incubation with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:10,000) (GE Healthcare Life sciences), membranes were washed with TBS-T and protein bands on the membrane were detected using an ECL-Plus immunoblotting chemiluminescence system (GE Healthcare Life sciences). Membranes were imaged using ImageQuant LAS 500TM camera (GE Healthcare Life sciences).

Cells transfected with shRNA were lysed in NP-40 lysis buffer (25 mM Tris, pH 7–8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100), and protein concentration in the cell lysate was quantified using a Bradford protein assay (Bio- Rad), as previously described^{30 (link)}. Protein samples were incubated at 100 °C for 5 min and electrophoresed on 10% SDS–polyacrylamide gels. Proteins were transferred to PVDF membranes (GE Healthcare Life Sciences). Membranes were blocked in 5% non-fat milk in Tris- buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Membranes were then incubated at 4 °C with following primary antibody rabbit anti-EHD1 (24657-1-AP, Proteintech), mouse anti-α-tubulin (T5168, Sigma) diluted in 5% non-fat milk overnight (1:500 and 1:10,000, respectively) then washed several times with TBS-T. After 1-h incubation with horseradish peroxidase-conjugated with respective IgG secondary antibody (1:10,000) (GE Healthcare Life Sciences), membranes were washed with TBS-T and protein bands on the membrane were detected using an ECL-Plus immunoblotting chemiluminescence system (GE Healthcare Life Sciences). Membranes were imaged using ImageQuant LAS 500 camera (GE Healthcare Life Sciences), as previously described^{30 (link)}.