Journal: Nature Communications
Article Title: SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors
Figure Lengend Snippet: Vps13B specifically binds to Rab14 and Rab6 and to PtdIns(3)P. a , b Association of endosomal Rab proteins with Vps13B, revealed by co-immunoprecipitation of endogenous Vps13B with GFP (or mRFP)-tagged Rab proteins. a GFP (or mRFP)-tagged proteins were precipitated from cell lysates using a GFP-trap or anti-mRFP antibody. Bound proteins were analyzed by immunoblotting for Vps13B, GFP, and mRFP. b Cross-linked cells were lysed and immunoprecipitated with anti-Vps13B antibody. The interaction of Rab proteins was detected and indicated Rab protein antibodies. c Coimmunoprecipitation of VPS13B with mRFP-Rab14-WT, GTP-locked mRFP-Rab14Q70L (QL), or GDP-locked mRFP-Rab14S25N (SN) expressed in HeLa cells. Anti-RFP antibody was used for immunoprecipitation of mRFP-tagged Rab14 proteins. There was no significant difference in the binding of Vps13B to the Rab14 variants. d Vps13B co-localizes with Rab6 and Rab14 upon expression in HeLa cells. Top panel: cells were transfected with mRFP-Rab14 and Vps13B-GFP and analyzed for the presence of GFP and mRFP, respectively, by fluorescence microscopy. Bottom panel: cells were transfected with GFP-Rab6. In this case, the distribution of endogenous Vps13B was monitored by immunocytochemistry. White arrows in the upper inserts show vesicles positive for both Vps13B and Rab14. Bottom panels show line scans on the lines in the upper panels and the percentage of colocalization of Rab14 with Vps13B. Scale bar, 10 µm. e PIP strip-binding assay of Vps13B. Perinuclear supernatant from Vps13B-GFP overexpressing cells was incubated with a membrane strip containing the phosphoinositides as indicated and probed for bound Vps13B using an anti-GFP antibody. f Binding of Rhodamine-labeled liposomes to Vps13B-GFP that was immobilized on magnetic Dynabeads. After incubation, the beads were washed, and bound liposomes were detected by Rhodamine fluorescence. Binding was only detectable when Vps13B-GFP was bound to the beads and when the liposomes contained 5% PtdIns(3)P. Error bars indicate S.E.M., *** P < 0.001, determined by unpaired t- test
Article Snippet: Primary antibodies used were obtained from the following companies: anti-APPL1 (3858), anti-LC3B (4599), and anti-Rab7 (9367) from Cell Signaling; anti-EEA1 (612006) and anti-GM130 (560257) from BD Biosciences; anti-M6PR (ab2733), anti-LAMP1 (ab24170), anti-mitofilin (ab110329), anti-mitofusion2 (ab101055), and anti-cathepsin D (ab6313) from Abcam; anti-Tfn receptor (sc-65882) from Santa Cruz Biotechnology; anti-LBPA (Z-PLBPA) from Echelon; anti-RFP (R10367), anti-Golgin97 (A-21270), anti-Vps52 (PA5–24408), and anti-Rab11 (71–5300) from Thermo Fischer Scientific; anti-PDI (700782) from ABfinity; anti-Vps51 (HPA061447), Vps13B (HPA043865) from Atlas antibodies; anti-Rab6 (10187–2-P) and anti-Rab35 (11329–2-AP) from Proteintech; anti-CD63 (H5C6) from Developmental Studies Hybridoma Bank; anti-Rab14 (R0656) from Sigma-Aldrich; anti-GFP (132002), anti-α-tubulin (302211; 1:10,000 dilution for Western blotting), anti-β-actin (251003; 1:10,000 dilution for Western blotting), anti-Rab5 (108011), anti-Stx6 (110062), anti-Stx13 (110132), anti-syntaxin 4 (110042), anti-syntaxin 16 (110162), and anti-VAMP4 (136002) from Synaptic Systems.
Techniques: Immunoprecipitation, Western Blot, Binding Assay, Expressing, Transfection, Fluorescence, Microscopy, Immunocytochemistry, Stripping Membranes, Incubation, Labeling