Journal: Molecular Cell

Article Title: Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames

doi: 10.1016/j.molcel.2023.01.023

Figure Lengend Snippet:

Article Snippet: After washing the cells, overexpressed PVT1-MP-V5 was stained with anti-V5 rabbit monoclonal antibody (1:500 in PBS with 5% BSA, #13202, Cell Signaling Technology), SRSF2-FLAG with anti-FLAG mouse monoclonal antibody (1:500 in PBS with 5% BSA, F1804, Sigma Aldrich), and SRSF6-BirA-Myc-His with anti-BirA chicken polyclonal antibody (1:500 in PBS with 5% BSA, BID-CP-100, BioFront Technologies) for 2 h at 4 °C.

Techniques: Transduction, Recombinant, Membrane, Protease Inhibitor, In Situ, Proximity Ligation Assay, Labeling, Luciferase, Phospho-proteomics, Knock-Out, RNA Sequencing, Sequencing, Microscopy, Immunofluorescence, Software, Targeted Proteomics

Journal: Scientific Reports

Article Title: Crystal structure of the Rab33B/Atg16L1 effector complex

doi: 10.1038/s41598-020-69637-0

Figure Lengend Snippet: Effects of Rab33B and Atg16 mutations on complex formation analyzed by GFP co-immunoprecipitation and Ni-sepharose pulldown experiments. (a) GFP Co-immunoprecipitation. Overexpression was done in HEK293 cells. Western blots were probed with either anti-GFP, anti-V5 or anti-LC3B antibodies. IP immunoprecipitation. ( b) Quantification of the western blot band intensities shown in (a) . Band intensities were calculated by normalizing the V5-IP blot band intensities against GFP-IP intensity. Error bars represent standard errors (SE) of three independent biological replicates. ( c) His-Rab33B(30–202) mutants were co-expressed with Atg16L1(153–210) wild-type and mutants. Samples were run on Schägger gels after elution from the Ni-Sepharose beads and then blotted onto nitrocellulose membranes. Membranes were probed with rabbit anti-Atg16L primary antibody and goat anti-rabbit IgG (HRP labeled) secondary antibody. Uncropped images of blots are shown in Figures S7 and S8.

Article Snippet: Afterwards the cells were blocked with 10% normal goat serum and 0.2% Triton-X100 in PBS for 1 h. The coverslips were inverted on top of a drop of 45–50 μL of specific antibodies (rabbit anti-V5 primary antibody (1:500) from GeneTex, mouse anti-WIPI2 (1:200) from Merck), and mouse Golgin-97 (1:200) from Thermo,) diluted in blocking buffer.

Techniques: Immunoprecipitation, Over Expression, Western Blot, Labeling

Journal: Scientific Reports

Article Title: Crystal structure of the Rab33B/Atg16L1 effector complex

doi: 10.1038/s41598-020-69637-0

Figure Lengend Snippet: Colocalization of EmGFP-Atg16L1, endo-WIPI2 and V5-Rab33B Q92L. (a) Endo-WIPI2b and V5-Rab33B Q92L were transiently expressed with EmGFP-Atg16L1 WT or mutants K198A, A202W or N206K in HeLa Cells. Arrows indicate colocalization, asterisks show no overlap. Scale bar, 10 µm. Figures are representative for three biological replicates. (b) Fraction of Atg16L1 overlapping with Rab33B Q92L as measured with Mander´s overlapping coefficient was analyzed using the ImageJ plugin JACoP.

Article Snippet: Afterwards the cells were blocked with 10% normal goat serum and 0.2% Triton-X100 in PBS for 1 h. The coverslips were inverted on top of a drop of 45–50 μL of specific antibodies (rabbit anti-V5 primary antibody (1:500) from GeneTex, mouse anti-WIPI2 (1:200) from Merck), and mouse Golgin-97 (1:200) from Thermo,) diluted in blocking buffer.

Techniques:

Journal: Biochimica et biophysica acta

Article Title: Involvement of activated SUMO-2 conjugation in cardiomyopathy.

doi: 10.1016/j.bbadis.2015.03.013

Figure Lengend Snippet: Fig. 6. SUMO targeted CAST for turnover. A. CAST was modified by SUMO. Western blot was performed on Hela cell lysates overexpressing V5-tagged CAST (CAST-V5) alone, or in the presence of either flag-epitoped SUMO-1-wt or SUMO-1-ΔGG, the latter which is the unconjugatable form of SUMO-1. Note that that a retarded species (SUMO-1 modified CAST, asterisk) was only detected in the presence of both CAST-V5 and SUMO-1-wt, but not in the presence of both CAST-V5 and SUMO-1-ΔGG. Upper panel: anti-V5 blot; lower panel: anti-flag blot. HMW, high molecular weight conjugates. B. CAST was a weak SUMO-2 target in Hela cells. Ni-NTA pulldown assay was performed on Hela cells transfected with 6× His tagged CAST- V5 alone, or in the presence of either SUMO-1 or SUMO-2, as indicated. The blot was probed with anti-V5 antibody. C. SUMO conjugation promoted CAST turnover. Western blot was per- formed on Hela cells expressing both CAST-V5 and SUMO-1-wt with or without cycloheximide (CHX) treatment (10 μg/ml) for 12 h as indicated. Note that the level of SUMO-conjugated CAST was significantly decreased after 12 h CHX treatment, while no obvious changes in the level of free CAST in these two groups. Asterisks indicate the sumoylated CAST. D. SUMO-fused CAST (SUMO-CAST) exhibited quick turnover. a, Schematic representation of SUMO-fused CAST-V5. b, Western blot was performed on Hela cell lysates expressing either CAST-V5 or SUMO-CAST-V5 in the presence or absence of CHX for 12 h, as indicated. Note that while CAST-V5 showed no significant changes in the protein levels after 12 h CHX treatment (compare lane 1 with lane 2, upper panel), SUMO-CAST-V5 showed a significant decrease at protein levels after CHX treatment (compare lane 4 with lane 3, upper panel). GAPDH served as a control. E. SUMO-CAST was cleaved by caspases. Hela cells were transfected with SUMO-CAST-V5 in the absence (lane 1) or the presence of CHX for 16 h. Various protease inhibitors with two dosages were added into the culture medium together with CHX from lane 3 to lane 10 as indicated. Note that only pan-caspase inhibitor was able to delay the turnover of SUMO- CAST-V5 (compare lanes 9 and 10 with lane 2 treated with vehicle). Upper panel: anti-V5; lower panel: anti-GAPDH-HRP as a control.

Article Snippet: Anti-V5-HRP, Annexin-VAlexa Fluor 594, Alexa Fluoro®488 anti-goat antibody, goat serum, Histostain-SP Kit (Invitrogen), anti-V5 antibody (Bethyl Laboratories, Inc.), anti-HA-HRP (Genscript), anti-GAPDH-HRP, anti-SUMO-1, anti-SUMO-2/3 and anti-Calpastatin antibodies (Santa Cruz), anti-Calpain 2 antibody (Cell Signaling Technology), anti-V5 agarose affinity gel (Sigma-Aldrich), bead-conjugated anti-SUMO-2/3 antibody (Santa Cruz), anti-mouse IgG agarose (Sigma-Aldrich), t-BOCLeu-Met-chloromethylaminocoumarin (t-BOC-LM-CMAC, Invitrogen), Ni-NTA agarose (Qiagen), RestoreWestern blot stripping buffer (Thermo Scientific Pierce), inhibitors for calpains 1 and 2, cathepsin, and caspases (pan caspase inhibitor Z-VAD(OM3)-FMK) (Santa Cruz). conjugation in cardiomyopathy, Biochim.

Techniques: Western Blot, High Molecular Weight, Transfection, Conjugation Assay, Expressing, Control

Journal: Biochimica et biophysica acta

Article Title: Involvement of activated SUMO-2 conjugation in cardiomyopathy.

doi: 10.1016/j.bbadis.2015.03.013

Figure Lengend Snippet: Fig. 7. SUMO fusion altered intracellular localization pattern of CAST. A, CAST-V5 or SUMO-fused CAST-V5 in Hela cells were visualized by immunostaining using anti-V5 antibody (green fluorescence). DAPI (blue fluorescence) was used for nuclear staining. Note the diffused pattern of CAST (left panel) and the aggregated pattern of SUMO-CAST (right panel) in cytoplasm. Bar indicates 50 μm. B, Bar graph indicates the percentage of cells with diffused (CAST-V5) or aggregated (SUMO-CAST-V5) localization, respectively. Data were collected from four inde- pendent experiments. **p b 0.001.

Article Snippet: Anti-V5-HRP, Annexin-VAlexa Fluor 594, Alexa Fluoro®488 anti-goat antibody, goat serum, Histostain-SP Kit (Invitrogen), anti-V5 antibody (Bethyl Laboratories, Inc.), anti-HA-HRP (Genscript), anti-GAPDH-HRP, anti-SUMO-1, anti-SUMO-2/3 and anti-Calpastatin antibodies (Santa Cruz), anti-Calpain 2 antibody (Cell Signaling Technology), anti-V5 agarose affinity gel (Sigma-Aldrich), bead-conjugated anti-SUMO-2/3 antibody (Santa Cruz), anti-mouse IgG agarose (Sigma-Aldrich), t-BOCLeu-Met-chloromethylaminocoumarin (t-BOC-LM-CMAC, Invitrogen), Ni-NTA agarose (Qiagen), RestoreWestern blot stripping buffer (Thermo Scientific Pierce), inhibitors for calpains 1 and 2, cathepsin, and caspases (pan caspase inhibitor Z-VAD(OM3)-FMK) (Santa Cruz). conjugation in cardiomyopathy, Biochim.

Techniques: Immunostaining, Staining