Skip to main content
. Author manuscript; available in PMC: 2010 Nov 7.
Published in final edited form as: Traffic. 2006 May;7(5):574–588. doi: 10.1111/j.1600-0854.2006.00410.x

Figure 2. Clathrin LC and Sla2p interact directly in a region containing the coiled-coil domain of Sla2p, but the HC terminal domain does not interact with Sla2p.

Figure 2

(A) Bacterially expressed GST fusions prebound to glutathione sepharose beads were used to pull down bacterially expressed 6xHis-Clc1p. GST-Bmh1p was used as a control for nonspecific binding. Blots were probed with indicated antibodies. (B) Bacterially expressed GST fusions prebound to glutathione sepharose beads were incubated with WT (SL1629) yeast extract to pull down clathrin HC. (C) YPJ96-4 was transformed with GBD bait plasmids pTMN9 (GBD-SLA2), pKH49 (GBD-sla2-292-968), pKH47 (GBD-sla2-292-501), pTMN38 (GBD-ENT1) or pGBDU (GBD empty) in combination with GAD prey plasmids pGAD (GAD empty) and pTMN8 (GAD-chc1-1-500). Equal numbers of cells from transformants were spotted on complete synthetic medium lacking leucine and uracil (C-LEU-URA) and medium lacking adenine, leucine and uracil (C-ADE-LEU-URA) and grown for 3 days. (D) Coomassie Blue-stained gel of fractions (31–47) from a Sephacryl S-1000 column derived from CCV purification of WT (SL1463) yeast. (E) Western blots of yeast extracts from WT + pSLA2 2μ (SL1463 + YEplac195-SLA2), WT (SL1463) and sla2Δ (HR1965) and fraction number 35 from S-1000 CCV preparation shown in D concentrated 50-fold. Blots were probed with αSla2p and αClc1p antibodies.