Atcc pcs 100 030

Human primary aortic smooth muscle cells (HASMCs, ATCC PCS-100-012) were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in vascular basal media (ATCC PCS-100-030) supplemented with vascular smooth muscle growth kit (ATCC PCS-100-042), containing 10% FCS, 10 U/mL penicillin, and 10 μg/mL streptomycin, at 37 °C under 5% CO₂. HASMCs were used between passages 3 and 6 and growth arrested with Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) containing 0.3% FBS overnight before the medium was changed to serum-free DMEM/F-12 one hour before the experiment. Cells were stimulated with Ang II (300 nM) for 2 and 24 h. The supernatant was collected, complemented with 10 µM EX527 and processed for immunoprecipitation similar to platelet supernatants. Cell lysates were washed with PBS and subjected to immunoprecipitation and Western blot as described for platelets.

The primary aortic smooth muscle cells (HASMC; ATCC PCS-100-012) were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in the vascular cell basal medium (ATCC PCS-100-030) with a vascular smooth muscle cell growth kit (ATCC PCS-100-042) at 37 °C in a 5% CO₂ incubator. The passage number of HASMC cells in the experiments was P7-P9.

Human vascular smooth muscle cells (hVSMC; primary aortic smooth muscle cells, ATCC-PCS-100-012) and HEK 293 T cells (ATCC-CRL-11268) were purchased from American Type Culture Collection. hVSMC were cultured in vascular basal media (ATCC PCS-100-030) supplemented with the vascular smooth muscle growth kit (ATCC PCS-100-042), 100 U/mL penicillin, 100 μg/mL streptomycin. hVSMCs were used between passages 2–5. HEK 293 T cells were cultured in high-glucose Dulbecco’s Modified’s Eagle Medium containing 10 mg/mL sodium pyruvate, 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The HTLA cell line, a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene^{18 (link)}, was generously provided by the laboratory of Dr. Bryan Roth and maintained in high glucose Dulbecco’s Modified’s Eagle Medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 µg/mL hygromycin B, and 2 µg/mL puromycin. All cells were cultured in a humidified environment at 37 °C, 5% CO₂.