Hek293 cells

HEK293 cells (ATCC) were maintained in DMEM supplemented with 10% FBS and
3.5 mg ml^−lglucose at
37 °C in 5% CO₂. Transfection was carried out
using lipofectamine (Life Technologies) according to the
manufacturer’s instructions. Twenty-four hours post transfection,
cells were washed and incubated for an additional 18 h in serum-free
growth media. siRNA (Qiagen) transfection was carried out as previously
described28 (link). Briefly, HEK293 cells that were
40–50% confluent in a six-well plate were transfected with the
plasmid encoding rat CB1 and
2.6 mg of siRNA (Qiagen) sequences targeting β-arrestin 1, β-arrestin 2 or non-silencing
RNA duplex for control. Silencing of β-arrestin 1 and β-arrestin 2 expression was
assessed by immunoblotting using anti-β-arrestin 1 (1:2,000; EMD Millipore, Billerica,
MA, USA) and anti-β-arrestin
2 (1:1,000; Novus Biologicals, Littleton, CO, USA)
antibodies, respectively. For the dominant-negative dynamin 2 experiments, the
dynamin 2 K44A mutant was
generated by site-directed mutagenesis (Stratagene, La Jolla, CA, USA) using the
human dynamin 2 cloned into
the pmCherryN1 vector (Addgene, Cambridge, MA, USA). HEK293 cells were
co-transfected with SEP-CB1R
and K44A dynamin 1-mCherry
cDNAs using lipofectamine described above.

HEK293 cells were from American Type Culture Collection (ATCC). Cells were mycoplasma negative. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 0.1 mg ml⁻¹ streptomycin and 100 U ml⁻¹ penicillin at 37 °C, 5% (vol/vol) CO₂. HEK293 cells were seeded at a density of 2.5 × 10⁵ cells per well onto six-well plates containing 24-mm round glass coverslips and allowed to grow for 24 h, after which they were transfected with the Effectene transfection kit (Qiagen) according to the manufacturer’s protocol.