Full-length cDNAs for RAPGEF2 and Bcl-2-associated X protein (BAX) were PCR-amplified from total human cDNAs and cloned into the pEGFP-C1 vector (Clontech, Mountain View, CA, USA) to generate pEGFP-RAPGEF2 and pEGFP-BAX. The E1357K mutation was introduced into pEGFP-RAPGEF2 by PCR-mediated mutagenesis to produce pEGFP-RAPGEF2-E1357K.
Primary human fibroblasts were established from punch biopsies on the forearm skin of the patient and a 43-year-old male control as described previously [23 (link)] and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 20% heat-inactivated (30 min, 55℃) fetal bovine serum (FBS), 1% non-essential amino acids, and antibiotics. Passage-matched control and patient fibroblasts (prior to passage 10) were used in each experiment. For inhibition of HDAC6, human skin fibroblasts were treated with 1 µM tubastatin A (Sigma-Aldrich, St. Louis, MI, USA) overnight at 37℃. Human HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and transfected using FuGENE HD transfection reagent (Promega, Madison, WI, USA).
Primary human fibroblasts were established from punch biopsies on the forearm skin of the patient and a 43-year-old male control as described previously [23 (link)] and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 20% heat-inactivated (30 min, 55℃) fetal bovine serum (FBS), 1% non-essential amino acids, and antibiotics. Passage-matched control and patient fibroblasts (prior to passage 10) were used in each experiment. For inhibition of HDAC6, human skin fibroblasts were treated with 1 µM tubastatin A (Sigma-Aldrich, St. Louis, MI, USA) overnight at 37℃. Human HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and transfected using FuGENE HD transfection reagent (Promega, Madison, WI, USA).
