3h leucine

Transdirect insect cell, an insect cell-free protein synthesis system, was obtained from Shimadzu (Kyoto, Japan). Human cDNAs (Flexi ORF clones) were purchased from Promega (Madison, WI, USA). [³H]leucine, [³H]myristic acid, and ECL prime western blotting detection reagent were from GE Healthcare (Buckinghamshire, UK). ENLIGHTNING was from PerkinElmer (Waltham, MA, USA). T7-Scribe standard RNA IVT kit was from CELLSCRIPT (Madison, WI, USA). The dye terminator cycle sequencing kit, Lipofectamine LTX and Plus reagent, MitoTracker Red CMXRos and Hoechst 33342 were from Life Technologies Corporation (Carlsbad, CA, USA). Anti-FLAG monoclonal antibody, anti-SAMM50 monoclonal antibody (WH0025813), anti-SAMM50 polyclonal antibody (HPA034537) and anti-Rabbit IgG-FITC antibody were from Sigma (St. Louis, MO, USA). Protein G-HRP conjugate was from Bio-Rad (Hercules, CA, USA). ImmunoStar LD was from Wako Pure Chemical (Osaka, Japan). X-ray film was from Eastman Kodak (Rochester, NY, USA). The other reagents used were from Wako Pure Chemical (Osaka, Japan), Daiichi Pure Chemicals (Tokyo, Japan) or Seikagaku Kogyo (Tokyo, Japan) and were of analytical or DNA grade.

Ricin B-chain was obtained from Vector Labs (Burlingame, CA), HEPES, α-lactose monohydrate, trypsin, pronase, bafilomycin A1, pepstatin A and CA074 methyl ester came from Sigma-Aldrich (St. Louis, MO). [³H]Leucine was purchased from GE Healthcare (Princeton, NJ), Na₂³⁵SO₄ and L-[³⁵S]Methionine came from Hartmann Analytic (Braunschweig, Germany). The mouse monoclonal anti-HA antibodies were obtained from Covance Research Products (Denver, CO), rabbit anti-BACE from Merck (Whitehouse Station, NJ), rabbit anti-Ricinus Communis-Lectin, and mouse anti-α-tubulin came from Sigma-Aldrich, whereas mouse monoclonal anti-ricin A-chain were purchased from Serotec (Oxford, UK). The mouse anti-calnexin were obtained from BD Biosciences (Palo Alto, CA), anti-calreticulin from BioSite (San Diego, CA). The rabbit anti-His antibodies came from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary anti-rabbit HRP and anti-mouse HRP antibodies, were obtained from Sigma-Aldrich, whereas anti-rabbit Alexa555 and anti-mouse Cy-3 were obtained from Jackson laboratories (Bar Harbour, MA).