Phrl sv40 vector

Cells were cultured in 24-well plates. All plasmids were prepared using a QIAGEN plasmid purification kit. Transient transfection was performed using Lipofectamine 3000 (Invitrogen), and the phRL-SV40 vector (Promega, Madison, WI, USA) was used as a transfection efficiency control. Forty-eight hours after transfection, the cells were lysed, and firefly and Renilla luciferase activities were evaluated using a dual-luciferase reporter assay system (Promega).

Cells were seeded into 24-well plates. All plasmids for transfection were isolated by QIAGEN plasmid purification kit (QIAGEN). Transient transfection by lipofectamine2000 (Invitrogen Carlsbad, CA, USA) were performed according to the manufacturer’s instruction and phRL-SV40 vector (Promega, Madison, WI, USA) was used as transfection efficiency control. Forty-eight hours after transfection, both firefly and renilla luciferase activities were measured using Dual-luciferase reporter assay system (Promega) with Luminoskan TL plus Luminometer (MTX, Labsystem, Vienna, VA, USA). The ratio of firefly’s activity to renilla was then obtained.

The BMP reporter (pGL3 BRE Luciferase) plasmid was a gift from Martine Roussel and Peter ten Dijkewas (Addgene, #45126)^{20 (link)}. The phRL-SV40 vector (Promega) was used as an internal control to normalize the variation in transfection efficiency. The wild-type Hela cell and two independent TMEM53 KO cell lines were used for luciferase assay. Cells were seeded in 24-well plates at a density of 5 × 10⁴ and cultured at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After 24 h, 100 ng/ml BMP2 (Humankine) was added into the medium, and the cells were transfected using TransIT-LT1 (Mirus Bio) according the manufacturer’s instructions. The luciferase activities were measured using the PicaGene Dual Sea Pansy Luminescence kit (Toyo Ink).