Ab167180

Cultured cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China). BCA kit (Pierce, Rockford, IL) was used to measure protein concentrations. The Flag and HA sequences were inserted into plasmids by the primers containing specific restriction sites. The plasmids were transformed into competent cells, screened, and amplified with LB containing ampicillin. High-concentration recombinant Flag-TMED3 and HA-FAM60A were obtained. These two plasmids were verified by DNA sequencing. Endotoxin-free extraction was carried out for subsequent cell transfection [18 (link)]. The equivalent amounts of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA). PVDF membrane was blocked with a blocking buffer and was incubated with an anti-TMED3 (ab223175, Abcam), anti-FAM60A (ab167180, Abcam), anti-GAPDH (AP0063, Bioworld, Nanjing, China), anti-DYKDDDDK Tag (14,793, Cell Signaling, Danvers, MA), anti-HA (ab9110, Abcam), anti-GAPDH antibody (AP0063, Bioworld), and the corresponding secondary antibodies, goat anti-rabbit IgG (A0208, Beyotime) or goat anti-mouse IgG (A0216, Beyotime). Immobilon Western Chemiluminescent HRP Substrate kit (Millipore) was used for color development and the bands were detected using an Amersham Imager 600 (GE Healthcare Little Chalfont, Buckinghamshire, UK).