Journal: eLife

Article Title: Building the vertebrate codex using the gene breaking protein trap library

doi: 10.7554/eLife.54572

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pGBT-RP2.1 , , RRID: Addgene_31828 , Genbank: HQ335170 , .

Techniques: Recombinant, Construct, Amplification, Reverse Transcription, Gel Extraction, Software, Microscopy

Journal: bioRxiv

Article Title: The ciliary targeting of membrane proteins by a ternary complex comprising transportin1, Rab8 and the ciliary targeting signal

doi: 10.1101/059451

Figure Lengend Snippet: (A) Bead-immobilized GST-f-CTS specifically pulled down GFP-Rab8-T22N but not -Q67L and -wt from the cell lysate expressing respective GFP chimeras. (B) Rabbit reticulocyte lysate from in vitro transcription and translation system contained endogenous TNPO1 but not Rab8. Rabbit reticulocyte lysate was mixed with methionine and Myc-TNPO1 or Myc-Rab8 expression DNA plasmids that contain T7 promoters for 1 hour at 30 °C. The lysate was subsequently separated by electrophoresis and blotted for Myc-tag, TNPO1 and Rab8. The Rab8 antibody used here should cross-react with rabbit Rab8 as the identity of antigen regions between human and rabbit is as high as 97%. (C) TNPO1 indirectly interacted with Rab8-T22N via f-CTS. The experiment also provided further evidence showing that Rab8-T22N increases the interaction between TNPO1 and f-CTS. HEK293T cell lysate triply co-expressing Myc-TNPO1, one of the GFP-Rab8 mutants (wt, Q67L and T22N) and CFF-HA or CD8aAcyto-HA was subjected to IP using anti-Myc antibody and co-IPed proteins were blotted for HA-tag and GFP. HA was blotted by HRP conjugated anti-HA antibody while GFP was blotted by anti-GFP primary antibody followed by HRP conjugated protein A. (D) The effect of Rab8 guanine nucleotide binding mutants on the ciliary localization of fibrocystin. Ciliated RPE1 cells co-expressing CFFAC-Myc and GFP, GFP-Rab8-wt, -Q67L or -T22N were subjected to surface labeling by CD8a antibody to reveal the ciliary localization of CFFAC-Myc. Cilia were identified by endogenous Arl13b staining. The cilium of interest is indicated by an arrow, enlarged and boxed at the upper right corner of each image. Scale bar, 10 μm. (E) RP2, rhodopsin or the CTS of prRDH assembled a ternary complex with Rab8-T22N and TNPO1. Bead-immobilized GST or GST-Rab8 mutants were incubated with HEK293T cell lysate expressing CFF-GFP (positive control), CD8a-prRDH-CTS-GFP, CD8a-GFP (negative control), rhodopsin-GFP or RP2-GFP. The bound GFP fusion proteins and endogenous TNPO1 were subsequently blotted. Note the high apparent molecular weight of full-length rhodopsin-GFP in our experimental condition (marked by “*”). (F) The screen showing that RP2 and CTSs of fibrocystin, prRDH and rhodopsin, but not peripherin, cystin, polycystin-1, polycystin-2, and SSTR3, demonstrate enhanced interaction with endogenous TNPO1 in the present of Rab8-GDP mutant. GST-CTSs immobilized on beads were incubated with HEK293T cell lysate expressing GFP-Rab8-Q67L or -T22N. The bound GFP-Rab8 and endogenous TNPO1 were subsequently blotted. CTSs of fibrocystin and prRDH were positive controls. Periph., peripherin. PC-1, polycystin-1. PC-2, polycystin-2. rhodo., rhodopsin. “*” denotes the specific band. In selected gel blots, numbers at the right indicate the molecular weight markers in kDa.

Article Snippet: To construct GST-RP2 , the CDS of RP2 was PCR amplified using Addgene plasmid #23636 (a gift from W. Hahn and D. Root) as the template and oligonucleotides 5’-GCA TCA GAA TTC ATG AAC AAG CAG TTC CGG-3’ and 5’-CCC GGG GTC GAC TAT TCC CAT CTG TAT ATC-3’ as primers.

Techniques: Expressing, In Vitro, Electrophoresis, Binding Assay, Labeling, Staining, Incubation, Positive Control, Negative Control, Molecular Weight, Mutagenesis

Journal: bioRxiv

Article Title: The ciliary targeting of membrane proteins by a ternary complex comprising transportin1, Rab8 and the ciliary targeting signal

doi: 10.1101/059451

Figure Lengend Snippet: (A) Rab8-T22N increased the binding of prRDH-CTS to TNPO1. HEK293T cell lysate expressing GFP-Rab8-wt, -Q67L or -T22N was subjected to pull-down by bead-immobilized GST-prRDH-CTS, GST-f-CTS (positive control) and GST (negative control) and the material pulled down was blotted for GFP-Rab8 and endogenous TNPO1. “*” denotes the band specific to GFP-Rab8. (B) Rab8-T22N increased the binding of rhodopsin-CTS and RP2 to TNPO1. HEK293T cell lysate co-expressing GFP-TNPO1 and Myc-Rab8-wt, -Q67L or -T22N was subjected to pull-down by bead-immobilized GST-f-CTS (positive control), GST-rhodopsin-CTS, GST-RP2 and GST (negative control) and the material pulled down was blotted for Myc-Rab8 and GFP-TNPO1. “*” denotes the corresponding GST-fusion protein used as the bait for the pulldown. (C) The interaction among RP2, Rab8-T22N and TNPO1 is abolished by C86Y and P95L mutation of RP2. HEK293T cell lysate expressing Myc-Rab8-wt, -Q67L or -T22N was subjected to pull-down by bead-immobilized GST-RP2, GST-RP2-C86Y,P95L and GST (negative control) and the material pulled down was blotted for Myc-Rab8 and endogenous TNPO1. “*” denotes the corresponding GST-fusion protein used as the bait for the pull-down. The intensity scaling of the same anti-GFP blot is adjusted to show both bright (intensity scaling 1) and weak bands (intensity scaling 2). In selected gel blots, numbers at the right indicate the molecular weight markers in kDa. (D) Model of ciliary targeting of fibrocystin. See the main text for the description.

Article Snippet: To construct GST-RP2 , the CDS of RP2 was PCR amplified using Addgene plasmid #23636 (a gift from W. Hahn and D. Root) as the template and oligonucleotides 5’-GCA TCA GAA TTC ATG AAC AAG CAG TTC CGG-3’ and 5’-CCC GGG GTC GAC TAT TCC CAT CTG TAT ATC-3’ as primers.

Techniques: Binding Assay, Expressing, Positive Control, Negative Control, Mutagenesis, Molecular Weight