Journal: Cell host & microbe

Article Title: Systematic identification of host cell regulators of Legionella pneumophila pathogenesis using a genome-wide CRISPR screen

doi: 10.1016/j.chom.2019.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-FLAG antibody (#14793, Cell Signaling Technology, 1:800 dilution), mouse monoclonal anti-FLAG antibody (F1804, Sigma-Aldrich, 1:800 dilution), mouse monoclonal anti-RABIF antibody (sc-390759, Santa Cruz Biotechnology, 1:1000 dilution), rabbit monoclonal anti-Rab10 antibody (#8127, Cell Signaling Technology, 1:600 dilution), rabbit polyclonal anti-Rab1A antibody (sc-311, Santa Cruz Biotechnology, 1:200 dilution), and mouse monoclonal anti-GAPDH antibody (AM4300, Thermo Fisher Scientific, 1:4000 dilution) were used for western blotting.

Techniques: Plasmid Preparation, Recombinant, Sequencing, Software

Journal: bioRxiv

Article Title: Ubiquitin Proteasome System Components, RAD23A and USP13, Modulate TDP-43 Solubility and Neuronal Toxicity

doi: 10.1101/2025.02.06.636899

Figure Lengend Snippet: HA-mTDP-43 or HA-WT-TDP-43 HEK293 cells were induced for 24 hours, collected and lysed. Protein lysates were immunoprecipitated (IP) with anti-HA antibody and immunoblotted for indicated proteins. IP fraction (25% loaded), input (30 ug loaded), and unbound (∼5% loaded). A. Immunoblot following IP of HA-mTDP-43 showing co-IP of USP13. B. Immunoblot following IP of HA-WT-TDP-43 showing co-IP of USP13. C. Immunoblot following IP of HA-mTDP-43 showing no co-IP of RAD23A. D. HA-mTDP-43 HEK293 cells transfected to knockdown USP13 and express myc-Ubiquitin, induced for 24 hours, collected and lysed. HA-mTDP-43 was immunoprecipitated. Immunoblot for myc-Ubiquitin shows no significant changes in abundance, indicating USP13 knockdown does not affect the ubiquitination of HA-mTDP-43. E. HA-mTDP-43 HEK293 cells transfected to knockdown USP13 , induced for 24 hours, lysates were collected and Biotin-TUBE reagent was added. Biotin-TUBE was immunoprecipitated. Immunoblot for HA-mTDP-43 shows no significant changes in abundance, indicating USP13 knockdown does not affect the ubiquitination of HA-mTDP-43. F, G. Inducible HA-mTDP-43 Q331K Flp-In T-REx HEK293 cells were plated, transfected with vector, FLAG-WT-USP13 or FLAG-USP13 C345A (catalytically inactive), induced with DOX for 24hrs, collected and subjected to SarkoSpin fractionation. (F) HA-mTDP-43 Q331K soluble and insoluble fractions and FLAG-USP13 abundance, representative immunoblot. (G) FLAG-WT-USP13 and FLAG-USP13 C345A significantly increases the percentage of HA-mTDP-43 in the insoluble fraction (quantified as a percent of total TDP-43) relative to negative control. Data represent the mean +/−SD, N=7, two technical replicates, one way ANOVA, F(2,36)=4.702, *p<0.05.

Article Snippet: The following antibodies were used in this study: RAD23A (Abcam; ab108592), RAD23A (Cell Signaling; 24555S), RAD23B (Abcam; ab86781), HA (Cell Signaling; C29F4), GAPDH (Sigma; G8795), Actin (Novus; NB600-501), USP13 (Proteintech; 16840-1-AP), TDP-43 (Proteintech; 10782-2-AP), ubiquitin (Enzo; BML-PW0930-0100), Myc (Cell Signaling; 2278S), FLAG (Cell Signaling; 2368S), PBXIP1 (Proteintech; 12102-1-AP), CHCHD10 (Proteintech; 25671-1-AP), NDUFS5 (Proteintech; 15224-1-AP)

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Transfection, Knockdown, Plasmid Preparation, Fractionation, Negative Control

Journal: Brain Pathology

Article Title: Alteration of Upstream Autophagy‐Related Proteins ( ULK1 , ULK2 , B eclin1, VPS34 and AMBRA1 ) in L ewy Body Disease

doi: 10.1111/bpa.12297

Figure Lengend Snippet: Time course analysis of upstream autophagosomal proteins. (A) Chronological upregulation of the proteins (ULK1, ULK2, VPS34, AMBRA1, p‐Beclin1 and Beclin1) parallels the accumulation of Lewy body (LB)‐like inclusions. LC3‐II is also upregulated relative to LC3‐I. Significant increase in the expression levels of ULK1 (B), ULK2 (C), Beclin1 (D), p‐Beclin1 (E) and VPS34 (F), but not AMBRA1 (G), 48 h after transfection. Values are expressed as means + standard deviation of three experiments. *P < 0.05, ** P < 0.01.

Article Snippet: Two other anti‐AMBRA1 antibodies (ProSci‐incorporated and Cell Signaling Technology) showing similar immunostaining of LBs. ( H ) Anti‐AMBRA1 antibody detecting a band of 150 kDa, corresponding to a band of FLAG‐AMBRA1.

Techniques: Expressing, Transfection, Standard Deviation

Journal: Brain Pathology

Article Title: Alteration of Upstream Autophagy‐Related Proteins ( ULK1 , ULK2 , B eclin1, VPS34 and AMBRA1 ) in L ewy Body Disease

doi: 10.1111/bpa.12297

Figure Lengend Snippet: Immunoreactivity of upstream autophagosomal proteins in human subjects. The neurons in the hippocampus of normal control subjects (A, D, G, J). Brainstem‐type Lewy bodies (LBs) in the substantia nigra in patients with Parkinson's disease (PD) (B, E, H, K) and cortical LBs in the temporal cortex of patients with dementia with LBs (DLB) (C, F, I, L). Weak immunoreactivity for ULK1 (A), ULK2 (D), VPS34 (G) and AMBRA1 (J) in the neuronal cytoplasm. The core of brainstem‐type LBs is moderately immunopositive for ULK1 (B), ULK2 (E) and VPS34 (H). Cortical LBs are slightly positive for ULK1 (C), ULK2 (F) and VPS34 (I). Intense immunoreactivity for AMBRA1 in the core of brainstem‐type LBs (K) and cortical LBs (L). Bars = 10 μm.

Article Snippet: Two other anti‐AMBRA1 antibodies (ProSci‐incorporated and Cell Signaling Technology) showing similar immunostaining of LBs. ( H ) Anti‐AMBRA1 antibody detecting a band of 150 kDa, corresponding to a band of FLAG‐AMBRA1.

Techniques:

Journal: Brain Pathology

Article Title: Alteration of Upstream Autophagy‐Related Proteins ( ULK1 , ULK2 , B eclin1, VPS34 and AMBRA1 ) in L ewy Body Disease

doi: 10.1111/bpa.12297

Figure Lengend Snippet: Double immunofluorescence staining of the substantia nigra in Parkinson's disease ( PD ). ULK1, ULK2, VPS34 and AMBRA1 appear green; phosphorylated α‐synuclein (p‐α‐Syn) appears red; and overlap of ULK1, ULK2, VPS34 or AMBRA1 and p‐α‐Syn appears yellow (merged). Immunoreactivity for ULK1, ULK2, VPS34 and AMBRA1 is more apparent in the core of Lewy bodies, whereas the peripheral portions show intense staining with anti‐p‐α‐Syn. Bars = 10 μm.

Article Snippet: Two other anti‐AMBRA1 antibodies (ProSci‐incorporated and Cell Signaling Technology) showing similar immunostaining of LBs. ( H ) Anti‐AMBRA1 antibody detecting a band of 150 kDa, corresponding to a band of FLAG‐AMBRA1.

Techniques: Double Immunofluorescence Staining, Staining

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Role of the di-proline motif in degradation of Δ9-desaturase. N-terminal amino acid sequences of Δ9-desaturases were compared (D. melanogaster DESAT1 (NP_652731), Mus musculus SCD1 (NP_033153), M. musculus SCD3 (NP_077770), Homo sapiens SCD1 (NP_005054), Danio rerio stearoyl-CoA desaturase (AAO25582), and S. cerevisiae Ole1 (CAA96757)) (A). Schematic illustration of DESAT1, mouse SCD1, and constructed mutants is shown (B). S2 cells expressing mouse SCD1-FLAGC, chimera-FLAGC, chimera (AA)-FLAGC, and SCD1 (di-Pro)-FLAGC were treated with C18:1 (100 μm) for 6 h (C) or DESAT1 inhibitor 37c (1 μm) for 16 h (D), and the amounts of endogenous DESAT1, FLAG-tagged exogenous Δ9-desaturases, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively. Band intensities were determined by ImageJ software, and levels of SCD1 proteins are shown relative to the amount of SCD1 protein in vehicle-treated cells (C and D). Mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Construct, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Identification of protease involved in the degradation of DESAT1. S2 cells were treated with calpeptin (50 μm), MG132 (50 μg/ml), or chloroquine (50 μm) for 6 h (A). S2 cells were treated with dsRNA against GFP, CalpA, CalpB, CalpC, or sol for 72 h and then exposed to C18:1 (100 μm) for 6 h (B). S2 cells expressing DESAT1-FLAGC and DESAT1 (P2A/P3A)-FLAGC were treated with calpeptin (50 μm) for 6 h (C). The amounts of endogenous DESAT1, FLAG-tagged exogenously expressed DESAT1, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively (A–C). Band intensities were determined by ImageJ software, and levels of DESAT1 proteins are shown relative to the amount of DESAT1 protein in vehicle-treated cells (A–C). Mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Expressing, Software