Pcmv6 ac gfp sim2

TE8 cells were plated at 2.5 × 10⁶ per 10‐cm dish and transiently transfected with either pCMV6‐AC‐GFP‐SIM2 or no insert of pCMV6‐neo (OriGene Technologies) by using Lipofectamine 2000 reagent (Invitrogen). Cells were treated with 5 μmol/L proteasome inhibitor, MG‐132 (Sigma‐Aldrich, St Louis, MO, USA) for 18 hours and, 48 hours after transfection, cells were collected and lysed in lysis buffer (20 mmol/L Tris‐HCl, 20% glycerol, 300 mmol/L KCl, 5 mmol/L 2‐mercaptoethanol, 1 mmol/L Pefabloc (Roche, Basel, Switzerland), and 25 μmol/L MG‐132. Immunoprecipitation was conducted using Immunoprecipitation Kit‐Dynabeads Protein G (Life Technologies, Rockville, MD, USA). Cell lysates were incubated with anti‐GFP antibody (OriGene Technologies) or control mouse IgG at 4°C overnight, and then incubated with 50 μL Dynabeads at 4°C for 4 hours. The Dynabeads‐antibody‐antigen complex was washed and resuspended in SDS sample buffer, then incubated at 95°C for 5 minutes. Eluted proteins were fractionated by SDS‐PAGE and detected by western blot.

pCMV6‐AC‐GFP containing the long isoform of the SIM2 cDNA and pCMV6‐neo containing SIM2s cDNA were purchased from OriGene Technologies (Rockville, MD, USA). Cells were plated at 2 × 10⁶ per 10‐cm dish, and transfected with either pCMV6‐AC‐GFP‐SIM2 or pCMV6‐SIM2s or no insert of pCMV6‐neo (OriGene Technologies) by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Forty‐eight hours later, cells were selected with G418 (0.4 mg/mL) for 1 month. Colonies were transferred to larger plates, and expression of different splicing isoforms of SIM2 mRNAs was examined by quantitative real‐time PCR.