Western blot was performed as per previous publications [17 (link)–19 (link)]. Cells were lysed by 200 μL RIPA lysis buffer (R0010, Solarbio) and then centrifuged with 12000 rpm for 4 minutes at 4° C. The supernatant was obtained to separate proteins. An BCA Protein Assay Kit (PC0020, Solarbio) was used to detect the concentration of total proteins.
Total proteins of the same concentration (30 μg) were loaded in 10% gel of SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (Millipore, MA, USA) and incubated overnight together with primary antibodies, including anti-SHOC2 (1:1000 dilution, ab229805, Abcam, MA, USA), anti-ERK1/2 (1:1000 dilution, ab184699, Abcam), anti-pERK1/2 (1:1000 dilution, ab65142, Abcam) and anti-β-actin (1:1000 dilution, ab8226, Abcam). Then, the membranes and goat antirabbit IgG H&L (HRP) secondary antibody (ab205718, Abcam) were incubated at room temperature for 1 hour. Then the membranes were immersed in 200 μL Immobilon Western Chemiluminescent HRP substrate (WBKLS0100, Millipore) and protein signals were recorded by the Bio-Rad ChemoDox XRS System (Bio-Rad Laboratories, CA, USA).
Total proteins of the same concentration (30 μg) were loaded in 10% gel of SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (Millipore, MA, USA) and incubated overnight together with primary antibodies, including anti-SHOC2 (1:1000 dilution, ab229805, Abcam, MA, USA), anti-ERK1/2 (1:1000 dilution, ab184699, Abcam), anti-pERK1/2 (1:1000 dilution, ab65142, Abcam) and anti-β-actin (1:1000 dilution, ab8226, Abcam). Then, the membranes and goat antirabbit IgG H&L (HRP) secondary antibody (ab205718, Abcam) were incubated at room temperature for 1 hour. Then the membranes were immersed in 200 μL Immobilon Western Chemiluminescent HRP substrate (WBKLS0100, Millipore) and protein signals were recorded by the Bio-Rad ChemoDox XRS System (Bio-Rad Laboratories, CA, USA).
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