Dyn2, dynamin 2; GFP, green fluorescent protein; GTP, guanosine triphosphate; PM,
plasma membrane; TfR, transferrin receptor. MiniPrep Express Matrix and Luria-Bertani medium were from BIO 101 (Vista, CA). Restriction enzymes were from New England Biolabs (Beverly, MA). Both anti-clathrin (X22) and anti-α-AP2 antibodies were collected from the supernatant of the X22 hybridoma and AP.6 hybridoma cell lines (ATCC, Rockville, MD). TfR1 antibody was purchased from Zymed Laboratories (San Francisco, CA). Anti-TfR1-N, pan-dynamin (MC63 and MC65), and Dyn2 antibodies were as described.14, 15 An anti-TfR2 antibody was raised against the peptide sequence QWSPRPSQTIYRRVEGPQLENLEEEDREEGE, corresponding to amino acids 13-43 in full-length rat TfR2 (Accession
number XM_222022). Transferrin and secondary antibodies conjugated LY2157299 to Alexa Fluor 594 or 488 were from Invitrogen (Eugene, OR). Unless otherwise stated, all other chemicals and reagents were from Sigma (St. Louis, MO). Two TfR isoform complementary DNA (cDNA0 sequences were from GenBank (Accession numbers TfR1 NM_022712; TfR2 XM_222022). The primers designed for TfR1 were TfR1 5′, GCCGCTGCATTGCGGACAGAGG AGGTGCTT and TfR1 3′, GCACAACCAGCTCAA GTCTAGAAACAGACTACCC; and for TfR2 were TfR2 5′, ATGGTCCAAGAAATCCAGAGACCTGTT GCTGAG and TfR2 3′, TCAAAAGTTATTGTC EMD 1214063 cost GATGTTCCAAACGTCGCCACT. Full-length cDNA was amplified using the XL PCR kit (Applied Biosystems, Branchburg, NJ). The polymerase chain reaction (PCR) cycle conditions were as follows: for TfR1, 94°C for 1 minute and 62°C for 5 minutes for 28 cycles, followed by 72°C for 7 minutes; for TfR2, 94°C for 1 minute and 65°C for 5 minutes for 28 cycles, followed by 72°C for 7 minutes. The PCR fragments were ligated into TA vector pCR3.1 (Invitrogen, Carlsbad, CA). WT Dyn2(aa) -GFP
was as described.13 Baf-A1 supplier Clone 9 and Hep3b2 cells (ATCC CRL-1439, Rockville, MD) were as described13; HepG2 and HuH-7 cells were as described.16, 17 These cells are widely utilized to study hepatocyte function, although they are not polarized and do not express most hepatocyte-specific proteins. Primary rat hepatocytes18 in Williams medium E supplemented with ITS, dexamethasone, 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin were incubated at 5% CO2 / 95% air at 37°C. Cells were cultured on 22-mm microscope cover slips for transfections and immunocytochemistry. Transfections were using the Lipofectamine 2000 Reagent kit (Invitrogen) with 1 μg plasmid DNA per transfection. The conditions for stable expression of TfR2 -pCR3.1 in Clone 9 cells are as described.13 Transferrin endocytosis assays were performed as described.