Journal: Blood

Article Title: Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis

doi: 10.1182/blood-2013-05-506212

Figure Lengend Snippet: p85 becomes ubiquitinated upon Epo stimulation. (A) γ2A/HA-EpoR/JAK2 cells transiently expressing p85 and Flag-tagged Ub were stimulated with Epo for 15 minutes. Immunoprecipitated p85 under nondenaturing conditions was blotted with the indicated antibodies. (B) Ubiquitinated p85 was also detected by immunoprecipitation using FK2 antibody–conjugated beads under denaturing conditions and immunoblotting for p85. (C) Epo induces endogenous p85 ubiquitination in primary Ter119− erythroid progenitor cells. FK2, anti-ubiquitinated proteins antibody; IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Antibodies were obtained from the following sources: mouse anti-HA (Covance); JAK2, phospho-JAK2, phospho-tyrosine antibody 4G10 (Millipore); actin and Flag (Sigma); streptavidin agarose (Thermo Scientific); streptavidin-HRP (Biolegend); Cbl (BD Biosciences); rabbit anti-HA, pY 731 Cbl, Alexa Fluor 488 conjugated anti-myc and glyceraldehyde 3-phosphate dehydrogenase (GAPDH); cell signaling; p85β and epsin-1 (Santa Cruz); anti-ubiquitinated proteins antibody FK2-conjugated beads (MBL); and T7 tag (Novagen).

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal: Blood

Article Title: Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis

doi: 10.1182/blood-2013-05-506212

Figure Lengend Snippet: Cbl ubiquitinates p85 in vitro. (A) T7-tagged p85 immunoprecipitated from HEK293T cells using anti-T7 antibody and Protein A beads was incubated with 200 ng of recombinant Cbl purified from BL21 in in vitro ubiquitination assay using recombinant E1, E2 (UbcH5B), and Flag-tagged wild-type Ub. After extensive washing, ubiquitinated p85 species were eluted from Protein A beads by SDS sample buffer and immunoblotted with the indicated antibodies. (B) p85 can be ubiquitinated at multiple sites. An in vitro ubiquitination assay was performed with a lysine-less ubiquitin mutant (KR) that cannot form ubiquitin chains. (C) p85 ubiquitination is lost in Cbl−/− MEFs and is restored in Cbl−/− MEFs reconstituted with Cbl.

Article Snippet: Antibodies were obtained from the following sources: mouse anti-HA (Covance); JAK2, phospho-JAK2, phospho-tyrosine antibody 4G10 (Millipore); actin and Flag (Sigma); streptavidin agarose (Thermo Scientific); streptavidin-HRP (Biolegend); Cbl (BD Biosciences); rabbit anti-HA, pY 731 Cbl, Alexa Fluor 488 conjugated anti-myc and glyceraldehyde 3-phosphate dehydrogenase (GAPDH); cell signaling; p85β and epsin-1 (Santa Cruz); anti-ubiquitinated proteins antibody FK2-conjugated beads (MBL); and T7 tag (Novagen).

Techniques: In Vitro, Immunoprecipitation, Incubation, Recombinant, Purification, Ubiquitin Assay, Mutagenesis

Journal: Autophagy

Article Title: Removal of hypersignaling endosomes by simaphagy

doi: 10.1080/15548627.2023.2267958

Figure Lengend Snippet: SQSTM1, NBR1 and LC3B are recruited to HGS-mutant endosomes. (a) Domain structure of APEX2-eGFP-HGS WT and APEX2-eGFP-HGS[ -770], which is missing the C-terminal clathrin box. Cell lines stably expressing APEX2-eGFP-HGS WT or -HGS[ -770] fusion proteins have been used for APEX2-proximity biotinylation of endosomally-recruited proteins. Volcano plot highlighting SQSTM1 and NBR1 enrichment in the APEX2-eGFP-HGS[ -770] mass spectrometry sample; FDR p-value 0.05. (b) Immunofluorescence staining of HGS, SQSTM1 and NBR1 in cells stably expressing eGFP-HGS WT or eGFP-HGS[ -770]. Cells are depleted for endogenous HGS or treated with control siRNA. The transgenic HGS WT and HGS[ -770] are siRNA stable. Top panel: SQSTM1 and NBR1 are recruited to HGS[ -770] endosomes (arrowheads indicating HGS, SQSTM1, NBR1 co-occurrence). Middle panel: LC3B is recruited with SQSTM1 and NBR1 to HGS[ -770] endosomes, but not in eGFP-HGS WT expressing cells (arrowheads). Bottom panel: HGS[ -770] endosomes display a strong ubiquitin (Ub) staining, which is not found in HGS WT or control cells. Scale bar: 10 µm; 5 µm for insets. (c) Representative electron micrographs of endosomes from HeLa cells stably expressing eGFP-HGS WT or HGS[ -770], depleted for endogenous HGS. Cells are stimulated 60 min with EGF to induce EGFR internalization. The 10-nm gold particles mark EGFRs. In eGFP-HGS WT cells degraded EGFR clusters in lysosomes (arrow). In eGFP-HGS[ -770] cells, EGFR is accumulating in a microdomain on the limiting membrane of endosomes (arrowheads). Bottom right panel shows an autophagosome containing a dysfunctional endosome. Scale bar: 250 nm.

Article Snippet: Mouse anti-ubiquitin (clone FK2, IF 1:400) was from EMD Merck Millipore (04-263). mouse anti-NBR1 (clone 4BR, IF 1:100) was from Santa Cruz Technology (sc-130380), mouse anti-NBR1 (6B11, WB 1:500 – 1:1000) was from Abnova (H00004077-M01).

Techniques: Mutagenesis, Stable Transfection, Expressing, Mass Spectrometry, Immunofluorescence, Staining, Transgenic Assay, Membrane

Journal: Autophagy

Article Title: Removal of hypersignaling endosomes by simaphagy

doi: 10.1080/15548627.2023.2267958

Figure Lengend Snippet: Loss of ESCRT-I subunits VPS37A and VPS37B leads to stalled simaphagy. (a) Representative immunofluorescence images of RPE-1 control or VPS37A KO cells. Loss of VPS37A leads to an increase in SQSTM1- and LC3B-positive objects and to an accumulation of ubiquitin (Ub) on endosomes. Below: western blot detecting full length VPS37A in RPE-1 control and VPS37A KO cells (asterisk indicates an unspecific background band detected by the VPS37A antibody). Quantification of a representative immunofluorescence experiment: sum fluorescence intensity of SQSTM1, LC3B and Ub objects show a significant increase in VPS37A KO cells. Mean ± SD of 7 images per condition and a total of 70-80 cells per condition. Two-tailed Student’s t test ****p<0.0001. Scale bar: 10 µm; 5 µm for insets. (B) Quantitative western blot analysis of EGFR degradation, after EGF stimulation in RPE-1 control cells, VPS37A KO cells and upon VPS37B knockdown. Left: representative western blot is shown. Degradation of EGFR is impaired upon VPS37A KO in combination with VPS37B knockdown. Right: western blot quantification showing residual EGFR after 15, 60, 120 or 180 min EGF stimulation. Values are displayed as percentage and normalized to the loading control and t = 15 min is set to 100%. Mean ± SD of three independent experiments. One-way ANOVA of 180 min timepoint *p < 0.1, **p < 0.01, ns = not statistically significant. (C) Movie stills of live-cell imaging experiments in VPS37A KO cells with additional knockdown of VPS37B. VPS37A KO cells stably expressing SNAP-LC3B and mCherry-SQSTM1 WT, were subjected to a 2 min pulse of EGF-Alexa647 before imaging. SNAP-LC3B was visualized by incubation with SNAP-OregonGreen ligand prior to imaging. Cells display simaphagy events (arrowheads), as well as an accumulation of phagophores not containing endosomes (asterisk). Right: Timeline of a representative simaphagy event shows recruitment of mCherry-SQSTM1 WT and SNAP-LC3B. Scale bar: 10 µm; 3 µm for insets I-IV; 2 µm for timeline.

Article Snippet: Mouse anti-ubiquitin (clone FK2, IF 1:400) was from EMD Merck Millipore (04-263). mouse anti-NBR1 (clone 4BR, IF 1:100) was from Santa Cruz Technology (sc-130380), mouse anti-NBR1 (6B11, WB 1:500 – 1:1000) was from Abnova (H00004077-M01).

Techniques: Immunofluorescence, Western Blot, Fluorescence, Two Tailed Test, Live Cell Imaging, Stable Transfection, Expressing, Imaging, Incubation

Journal: Autophagy

Article Title: Removal of hypersignaling endosomes by simaphagy

doi: 10.1080/15548627.2023.2267958

Figure Lengend Snippet: Simaphagy controls directed cell migration and can be detected in vivo . (a) Representative phase contrast images of RPE-1 control cells, VPS37A KO cells with and without knockdown of VPS37B using siRNA. Knockout of VPS37A leads to a more elongated cell morphology, a phenotype that is enhanced by knockdown of VPS37B. Scale bar: 100 µm. (b) Quantification of random migration patterns in RPE-1 control and VPS37A KO cells with and without VPS37B knockdown. 40-55 cells have been tracked for each condition over an imaging period of 18 h, with 20 min imaging intervals. Velocity, accumulative- and Euclidean distance are shown for each condition. The proliferation rate was manually scored for the imaging time of 18 h. One-way ANOVA *p < 0.05, **p <0.001, ns = not statistically significant. (c) Representative images and quantifications of wound healing assays performed with RPE-1 control and VPS37A KO cells with and without knockdown of VPS37B. VPS37A KO cells have an elevated migratory potential. This is further increased by a knockdown of VPS37B. Wound is shown at 1 h and 18 h after wounding (white line indicating cell front at t 0 = 1 h). Scale bar: 300 µm. Average wound width over time and quantification of wound width at 12 h and 15 h are shown. Quantifications from 3 areas per condition. One-way ANOVA *p < 0.05, **p <0.001, ns = not statistically significant. (d) Representative immunofluorescence staining in Drosophila eye-antennal discs. Left: Clusters of Hrs 28D cells (marked by nuclear RFP) display enlarged Rab5-positive endosomes with accumulating ubiquitin. The ref(2)P (middle panel) and Atg8a (right panel) proteins are recruited to ubiquitin-labelled enlarged endosomes in HRS 28D larvae. Scale bar: 10 µm, insets 5 µm.

Article Snippet: Mouse anti-ubiquitin (clone FK2, IF 1:400) was from EMD Merck Millipore (04-263). mouse anti-NBR1 (clone 4BR, IF 1:100) was from Santa Cruz Technology (sc-130380), mouse anti-NBR1 (6B11, WB 1:500 – 1:1000) was from Abnova (H00004077-M01).

Techniques: Migration, In Vivo, Knock-Out, Imaging, Immunofluorescence, Staining

Journal: Developmental Cell

Article Title: Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform

doi: 10.1016/j.devcel.2019.06.016

Figure Lengend Snippet:

Article Snippet: Agrose beads cross linked to anti-ubiquitin antibody FK2 , MBL , D058-8; RRID: AB_843667.

Techniques: Recombinant, Software

Journal: Journal of Cell Science

Article Title: An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

doi: 10.1242/jcs.222034

Figure Lengend Snippet: Aberrant vesicular structures containing GFP–VAMP7-R150E are deficient in typical regulators of intracellular trafficking. (A–C) Transgenic X. laevis expressing the GFP–VAMP7-R150E mutant and examined by CLEM. Retinas were first examined by confocal microscopy (A) and then processed and analyzed by EM (B). Six cells in the confocal optical section (a–f) were matched to the cells (a–f) in the EM micrograph. The asterisk marks a control photoreceptor (f), not expressing GFP–VAMP7-R150E. The boxed area in B is magnified in C. m, mitochondria, Ly, lysosome. (D–F) Transgenic retinas expressing GFP–VAMP7-R150E fusion protein (green) labeled with anti-LAMP1 (r) (D), anti-GFP (r) (E), or anti-βCOP (r) (F). Arrows point to localization of the proteins examined. (G) PLA between: Rab6 (r) and anti-GFP (m) detecting GFP–VAMP7-R150E. (H,I) EM of the photoreceptor cell expressing the R150E mutant (H). The boxed area is magnified in I. Arrows point to vesicular structures with electron-dense content. (J–M) Transgenic GFP–VAMP7-R150E retinas labeled with anti-IRBP (m) (J), anti-ASAP1 (r) (K), anti-FIP3 (r) (L) and anti-VARP (r) (M). (N) PLA between syntaxin 3 (r) and anti-GFP (m). (O) Retinas labeled with anti-peripherin (m) (red). (P,Q) The f1 generation of GFP–VAMP7-R150E-expressing retinas stained with WGA (red). (R) Labeling with anti-LC3 (r) (red, arrows). (S) Epithelial cells outside of Xenopus eye labeled with anti-LC3 (r) (red, arrows point to autophagosomes). (T,U) Labeling with anti-ATG16L1 (m) (red, arrow) (T), or anti-ubiquitin (m) (U). Cells were visualized by DIC. (V) The f1 generation of GFP-VAMP7-Y45E-expressing retinas stained with WGA (red). Scale bar: 25 µm (for A); 5 µm (for B,D–H); 10 µm (for J–N); 1 µm (for C,I); 12 µm (for O,R,S,T,U); 20 µm (for Q); 50 µm (for P,V). M, myoid region; E, ellipsoid region; G, Golgi, N, nucleus; m, mouse antibody; r, rabbit antibody.

Article Snippet: Mouse monoclonal antibodies: anti-SNAP-25 (SMI-81) ( Mazelova et al., 2009b ) (a gift from Michael C. Wilson, and 836303, BioLegend), anti-Rab11 (610656, BD Biosciences), anti-rhodopsin (11D5) ( Deretic and Papermaster, 1991 ), anti-VAMP7 (MAB6117, R&D Systems), anti-GM130 (11308-1-AP, BD Transduction Laboratories), anti-peripherin 5A11 ( Loewen et al., 2003 ), anti-ubiquitin (FK2, D058-3, Biomol), anti-IRBP ( Hessler et al., 1996 ) (a gift from Federico Gonzalez-Fernandez, University of Mississippi, Jackson, MS), anti-GFP (ab38689, Abcam) and mouse polyclonal anti-ATG16L1 (SAB1407607, Sigma).

Techniques: Transgenic Assay, Expressing, Mutagenesis, Confocal Microscopy, Labeling, Staining

Journal: PLoS ONE

Article Title: Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

doi: 10.1371/journal.pone.0013465

Figure Lengend Snippet: (A) Schematized overview of proteomic screen for the isolation and identification of synaptic ubiquitinated proteins from mice expressing a hexahistidine-tagged ubiquitin GFP fusion transgene under the human UbC promoter (Tg-Ub mice). (B) Representative immunoblot depicting the ubiquitin immunoreactivity in Tg-Ub and WT (non-Tg) P2′ synaptosomal membrane total lysates or following nickel-affinity purification. (C) Adult rate brain P2′ synaptosomal membranes were solubilized and then immunoprecipitated with either α-STIM1 or α-ubiquitin (FK2). Immune complexes were resolved by SDS-PAGE and subjected to western blot analysis with α-STIM1 (top) or α-ubiquitin (bottom) antibodies Arrow indicates the relative mobility of unmodified STIM1. (D) HA-ubiquitin and STIM1-GFP were co-expressed in HEK293 cells. The resulting lysates were subjected to immunoprecipitation with either α-GFP or control serum (normal rabbit serum, NRS). Arrow indicates the relative mobility of monoubiquitinated-STIM1GFP. Representative blots depicted from 2 to 3 independent experiments for C and D.

Article Snippet: FK2, α-ubiquitin conjugate (mAb, 1∶1000) antibody was purchased from Biomol (Plymouth Meeting, PA); α-ubiquitin (pAb, 1∶2000) antibody was purchased from Dakocytomation (Kyoto, Japan); α-synapsin antibody (pAb, 1∶1000) was purchased from Chemicon (Temecula, CA); α-PSD-95 antibody (mAb, 1∶1000) was purchased from Calbiochem (La Jolla, CA); α-GluR1 (pAb, 1∶1000 immunoblot) was purchased from Upstate Biotechnologies (Lake Placid, NY); α-STIM1 (mAb, 1∶1000 immunoblot; 1∶200 immunofluorescence) was purchased from BD Biosciences (Franklin Lakes, NJ); α-Calreticulin (pAb, 1∶1000) was purchased from StressGen (Victoria, BC, Canada); MAP2 (pAb, 1∶5000) was purchased from Abcam (Cambridge, UK).

Techniques: Isolation, Expressing, Ubiquitin Proteomics, Western Blot, Membrane, Affinity Purification, Immunoprecipitation, SDS Page, Control