Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: V5-antibody conjugated magnetic beads (MBL), were washed 3x with 1X PBS+0.1% Tween 20 (PBS-T) before combining it with lysate in a 1μL:2 μg ratio (ex.

Techniques: Recombinant, Transfection, Purification, Modification, Lysis, Blocking Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Expressing, Clone Assay, Plasmid Preparation, Sequencing, Immunoprecipitation, RNA Sequencing Assay, Mass Spectrometry, Software, Imaging, Microscopy, Magnetic Beads, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) SKNAS cells expressing V5-tagged KIF5AΔExon27 show increased microtubule (MT) co-localization compared with KIF5AWT as demonstrated by V5-KIF5A highlighting the MT tracks. Examples of KIF5A (V5; green) and β-tubulin (red) co-localization are indicated by arrowheads. Many cells have KIF5AΔExon27-associated MTs with a non-radial pattern (asterisks). Scale bars, 10 mm (wide view), 5 mm (enlargement).

Article Snippet: V5-antibody conjugated magnetic beads (MBL), were washed 3x with 1X PBS+0.1% Tween 20 (PBS-T) before combining it with lysate in a 1μL:2 μg ratio (ex.

Techniques: Expressing

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) Mass spectrometry analysis of V5-tagged KIF5AWT and KIF5AΔExon27 bound proteins in SKNAS cells. Venn diagram indicates the number of protein binding partners altered in KIF5AΔExon27 mutant immunoprecipitations. Yellow region: proteins that are unique to, or have ≥4× increase in the amount bound to, KIF5AΔExon27. Red region: proteins that are absent from, or have ≥4× decrease in the amount bound to, KIF5AΔExon27. Orange region: proteins that show no binding preference to either form of KIF5A.

Article Snippet: V5-antibody conjugated magnetic beads (MBL), were washed 3x with 1X PBS+0.1% Tween 20 (PBS-T) before combining it with lysate in a 1μL:2 μg ratio (ex.

Techniques: Mass Spectrometry, Protein Binding, Mutagenesis, Binding Assay

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: V5-antibody conjugated magnetic beads (MBL), were washed 3x with 1X PBS+0.1% Tween 20 (PBS-T) before combining it with lysate in a 1μL:2 μg ratio (ex.

Techniques: Recombinant, Transfection, Purification, Modification, Lysis, Blocking Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Expressing, Clone Assay, Plasmid Preparation, Sequencing, Immunoprecipitation, RNA Sequencing Assay, Mass Spectrometry, Software, Imaging, Microscopy, Magnetic Beads, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

doi: 10.1093/nar/gkaa564

Figure Lengend Snippet: The CASC3 N-terminus promotes but is not necessary to elicit NMD. ( A ) Schematic depiction of the TPI-4MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). ( B ) Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). ( C ) Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1–480 has a C-terminal deletion, whereas in the construct 110–480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). ( D ) Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure . Rescue protein expression is confirmed in E and F. Individual data points and means are plotted from n = 3 experiments. (E and F) Western blot of samples shown in Figure . The expression of rescue proteins was confirmed by an antibody against CASC3 ( E ) and an antibody recognizing the V5 tag ( F ).

Article Snippet: The following antibodies were used: anti-CASC3 amino acid residues 653–703 (Bethyl Laboratories, #A302-472A-M), anti-CASC3 amino acid residues 367–470 (Atlas Antibodies, #HPA024592), anti-EIF4A3 (Genscript), anti-FLAG (Cell Signaling Technology, #14793), anti-MAGOH (Santa Cruz Biotechnology, #sc-271365), anti-PABPC1 (Cell Signaling Technology, #4992), anti-RBM8A (Atlas Antibodies, #HPA018403), anti-SMG6 (Abcam, #ab87539), anti-SMG7 (Elabscience, #E-AB-32926), anti-Tubulin (Sigma-Aldrich, #T6074), anti-UPF3B (antiserum was raised in rabbits by Eurogentech against a C-terminal fragment of UPF3B (300–483) and affinity purified), anti-V5 (QED Bioscience, #18870), anti-XRN1 (Bethyl Laboratories, #A300-443A), anti-rabbit-HRP (Jackson ImmunoResearch, #111-035-006), anti-mouse-HRP (Jackson ImmunoResearch, #115-035-003).

Techniques: Binding Assay, Northern Blot, Stable Transfection, Construct, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

doi: 10.1038/srep30955

Figure Lengend Snippet: Characterization of candidates for ERAD E3 ligase ( a ) Subcellular localisation of E3 ligases. COS-1 cells stably expressing E3-V5 were subjected to immunofluorescence staining with anti-V5 and -PDI antibodies. ( b ) In vitro autoubiquitination assay. The E3 proteins produced by a transcription/translation system were immunoprecipitated with anti-V5 antibody, and then mixed in the reaction buffer with E1 (GST-tagged), E2 (GST-UbcH5c) and HA-ubiquitin. The reaction mixture was again immunoprecipitated with anti-V5 antibody and analysed via Western blotting using anti-ubiquitin or V5-antibodies. CS mutants defective in E3 activity were constructed by replacement of conserved coordinating Cys with Ser residues in the RING. Asterisk indicates the heavy chain of immunoglobulin. ( c ) E3 ligases protect against ER stress-induced cell death. Neuro-2a (N2a) cells stably expressing WT or ΔRING (ΔR) mutant of E3 ligases were transiently treated with Tm (1 μg/ml) or staurosporine (STS; 0.1 μM) and incubated for 48 h. The cells were stained with crystal violet. The eluted dye at an optical density of 590 nm was measured. Cell viability was calculated as follows: OD for assay/OD for vehicle control (0.1% dimethyl sulfoxide) well. The results are expressed as the fold increase compared with mock cells, in terms of means ± SD (three independent experiments in duplicate). Statistical analysis was performed using ANOVA followed by Bonferroni correction (vs. controls; *p < 0.05, **p < 0.01)

Article Snippet: The reaction products containing V5-tagged E3s were immunoprecipitated with anti-V5 antibody, and then mixed with Recombinant Human GST-Ubiquitin E1 Enzyme (UBE1) Protein (100 ng), Recombinant Human GST-UbcH5c/UBE2D3 Protein (100 ng), and Recombinant Human HA-Ubiquitin Protein (10 μg) purchased from Boston Biochem (Cambridge, MA) in a reaction buffer [100 μl, containing 40 mM Tris-HCl (pH 7.6), 5 mM MgCl 2 , 2 mM ATP and 2 mM dithiothreitol].

Techniques: Stable Transfection, Expressing, Immunofluorescence, Staining, In Vitro, Produced, Immunoprecipitation, Western Blot, Activity Assay, Construct, Mutagenesis, Incubation