Journal:

Article Title: Essential Role of hIST1 in Cytokinesis

doi: 10.1091/mbc.E08-05-0474

Figure Lengend Snippet: hIST1 binds to CHMP1A, CHMP1B, VPS4, and LIP5. (A) Sequence alignment showing the high degree of similarity between IST1 genes from different organisms. The shading on the alignment indicates conserved amino acids. (B) hIST1 fused to the VP16 activation domain was tested for interactions with the human components of ESCRT-I, -II, -III, and ESCRT-III–associated proteins by yeast two-hybrid assays. Error bars indicate the SD from the mean of triplicate measurements. (C) Coprecipitation assays showing binding of hIST1 to CHMP1A, CHMP1B, CHMP2A, VPS4, and LIP5. One percent of the starting cell lysate (INPUT) and 10% of the volume eluted from the beads were analyzed by Western blot with α-GFP antibody.

Article Snippet: The blots were sequentially probed with monoclonal antibodies α-HIV-1 p24 (183-H12-5C), α-GFP (Roche Diagnostics), or α-TSG101 (4A10; Abcam, Cambridge, United Kingdom), rabbit polyclonal antibodies α-IST1 (ProteinTech Group, Chicago, IL; catalog no. 51002-1-Ap), α-CEP55 (A01; Abnova, Heidelberg, Germany), or α-HSP90 (Santa Cruz Biotechnology) and with a peroxidase-conjugated antibody against mouse (Cell Signaling Technology) or rabbit (Cell Signaling Technology) and developed using chemiluminescent substrate reagents (Pierce Chemical, Rockford, IL).

Techniques: Sequencing, Activation Assay, Binding Assay, Western Blot

Journal:

Article Title: Essential Role of hIST1 in Cytokinesis

doi: 10.1091/mbc.E08-05-0474

Figure Lengend Snippet: (A) Mapping by yeast two-hybrid analysis of the regions needed in CHMP1B for interaction with IST1. Binding to CHMP1A was used as a positive control; the regions of VPS4 required for interaction with IST1. LIP5 was used as a positive control (B); and the regions of IST1 required for interaction with CHMP1A, CHMP1B, VPS4, or LIP5 (C). Error bars indicate the SD from the mean of triplicate measurements. (D) Micrographs showing that endogenous hIST1 is recruited to aberrant endosomes in the presence of YFP-VPS4 E228Q. Left, images acquired with YFP (pseudocolored in green); middle, images acquired with Alexa594 (pseudocolored in red); and right, overlaid images. A higher magnification of the boxed areas is shown in all panels and DNA is shown in blue. Bar, 10 μm.

Article Snippet: The blots were sequentially probed with monoclonal antibodies α-HIV-1 p24 (183-H12-5C), α-GFP (Roche Diagnostics), or α-TSG101 (4A10; Abcam, Cambridge, United Kingdom), rabbit polyclonal antibodies α-IST1 (ProteinTech Group, Chicago, IL; catalog no. 51002-1-Ap), α-CEP55 (A01; Abnova, Heidelberg, Germany), or α-HSP90 (Santa Cruz Biotechnology) and with a peroxidase-conjugated antibody against mouse (Cell Signaling Technology) or rabbit (Cell Signaling Technology) and developed using chemiluminescent substrate reagents (Pierce Chemical, Rockford, IL).

Techniques: Binding Assay, Positive Control

Journal:

Article Title: Essential Role of hIST1 in Cytokinesis

doi: 10.1091/mbc.E08-05-0474

Figure Lengend Snippet: IST1 contains a functional MIM. (A) Sequence alignment of the MIMs from the indicated human ESCRT-III proteins and the human and yeast IST1 genes. Positions of the six MIM residues contacting Vps4 MIT domain are indicated on top of the alignment and conserved amino acids are shaded in gray. (B) Coprecipitation assays showing binding of the indicated MIT containing proteins to YFP-CHMP1B or YFP-hIST1. One percent of the starting cell lysate (INPUT) and 10% of the volume eluted from the beads were analyzed by Western blot with α-GFP antibody. (C) hIST1 residues in MIM's position +2 and +3 were mutated (hIST1 L375A/K376A), and interactions with ESCRT-III and MIT-containing proteins were monitored using yeast two-hybrid assays. Error bars indicate the SD from the mean of triplicate measurements. (D) Cell lysates from bacteria expressing CHMP1B's MIM (GST-CHMP1B 180-196) or hIST1's MIM (GST-hIST1 363-379) were tested by coprecipitation assays for binding to the indicated MIT-containing proteins fused to YFP and binding was monitored with α-GFP mAb (WBαYFP). Bottom, GST fusion protein expression in cell lysates with Coomassie staining. (E) Schematic representation of CHMP1B and hIST1 proteins, showing the regions needed for their interaction (arrows), and the position of their MIMs.

Article Snippet: The blots were sequentially probed with monoclonal antibodies α-HIV-1 p24 (183-H12-5C), α-GFP (Roche Diagnostics), or α-TSG101 (4A10; Abcam, Cambridge, United Kingdom), rabbit polyclonal antibodies α-IST1 (ProteinTech Group, Chicago, IL; catalog no. 51002-1-Ap), α-CEP55 (A01; Abnova, Heidelberg, Germany), or α-HSP90 (Santa Cruz Biotechnology) and with a peroxidase-conjugated antibody against mouse (Cell Signaling Technology) or rabbit (Cell Signaling Technology) and developed using chemiluminescent substrate reagents (Pierce Chemical, Rockford, IL).

Techniques: Functional Assay, Sequencing, Binding Assay, Western Blot, Bacteria, Expressing, Staining