Hemagglutinin ha antibody

Geneticin (G418) and Lipofectamine 2000 were purchased from Invitrogen/Thermo Fisher Scientific (Waltham, MA, United States). Antibodies: The β_1/2-arrestin antibodies, hemagglutinin (HA) antibody, Anti-p-ERK antibody and Anti-ERK antibody were obtained from Cell Signaling Technology (Danvers, MA, United States). The rabbit Anti-APJ Receptor antibody was obtained Abcam (Cambridge, United Kingdom). The β-actin antibody and secondary antibodies were purchased from ZSGB Bio (Beijing, China).

HEK293 cells (2X10⁵ in 6 well plates) were transfected with MR-HA and HDAC-Flag plasmids by using Superfect reagents (Qiagen, Hilden, Germany) according to manufacturer’s recommendation. After 48 h, cells were stimulated with Aldo with or without pretreatment with inhibitors for 6h. Cell were lysed by a lysis buffer (20 mmol/L Tris, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1x proteinase inhibitor cocktail). The cell lysates (100 μg) were precleared with protein G agarose at 4°C for 2 h. After centrifugation (3000 g for 2 min), supernatants were incubated with 1μg of hemagglutinin (HA) antibody (Cell Signaling Technology, Beverly, MA) at 4°C for overnight. The immunocomplexes were washed with the lysis buffer (three times), and boiled after adding 2X protein sample buffer. To detect Flag tagged HDAC, Western blotting was used as described previously [1 (link)].