Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Time course of DR6-induced apoptosis. After induction of DR6 expression by the addition of tetracycline (Tet), cells were harvested at various time points as indicated and divided into two portions. Half of the harvested cells were directly lysed and separated by SDS-PAGE, followed by Western blot analysis. The other half of the cells were used for preparation of cytosolic extracts and mitochondria-containing membrane fraction as described under “Experimental Procedures.” A, top panel shows induction of DR6 expression. Second panel shows PARP cleavage. Third, fourth, fifth, and sixth panels show activation of caspase-3, caspase-9, caspase-8, and caspase-7, respectively. The activation of caspases was determined by the formation of active forms of these caspases. This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (bottom panel). B, top panels show release of cytochrome c from mitochondria (right column) to the cytosol (left column). Second panels show translocation of Bax from cytosol (left panel) to mitochondria (right panel). Third panel show tBid formation and translocation to mitochondria. Fourth panel shows the reprobe of the second panels with anti-COX I antibody confirming the mitochondria remained intact during preparation.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Expressing, SDS Page, Western Blot, Membrane, Activation Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Overexpression of antiapoptotic protein Bcl-2 and Bcl-xL inhibited DR6-induced apoptosis. Cell lysis and fractionation were performed as described previously. A and B, after 24-h transfection, a significant amount of Bcl-2 and Bcl-xL was detected (lanes 3 and 4, top panel). Because the recombinant Bcl-2 and Bcl-xL were expressed as a FLAG-tagged protein, the recombinant Bcl-2 and Bcl-xL were detected with a slower migration rate than endogenous Bcl-2 and Bcl-xL (compare lanes 3 and 4 with lanes 1 and 2). This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (fourth panel). Second and third panels show tetracycline (Tet)-induced DR6 expression, respectively; fifth and sixth panels, cytochrome c release from mitochondria; seventh and eighth panels, Bax translocation from cytosol to mitochondria. C, top panel shows Western blot for PARP; second to sixth panels, Western blots for caspases; seventh panel, Western blot for actin to indicate relative loading of lysate samples; eighth and ninth panels, Western blots for Bid; tenth and eleventh panels were stained for mitochondrial protein COX I.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Over Expression, Lysis, Fractionation, Transfection, Recombinant, Migration, Membrane, Expressing, Translocation Assay, Western Blot, Staining

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expression tests for ejection proteins gp14, gp15, and gp16 (A) Diagram of plasmids for gp14, gp15, and gp16. (B) Diagram of transformed colonies in different expression strains growing on agar plates treated with ampicillin (100 μg/mL). (C) Diagram of 100 mL starter cultures treated with ampicillin for different expression strains in 250 mL Erlenmeyer flasks. (D) Diagram of 1 L LB cultures treated with ampicillin and inoculated 1:50 with starter culture for different expression strains. (E–G) SDS-PAGE analysis for gp14, gp15, and gp16 before/after (−/+) induction with IPTG at OD600 = 0.6 and expression. Expected molecular weights are gp14, 21 kDa, gp15, 87 kDa and gp16, 146 kDa as incdicated on the SDS-PAGE gels in cyan, magenta, and green, respectively.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Expressing, Transformation Assay, SDS Page

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Workflow for the reconstitution of gp15:gp16 periplasmic tunnel of the T7 DNA-ejectosome.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques:

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection proteins gp15 and gp16 (A) SDS-PAGE results for sonicated samples for gp15 and gp16. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation. (B) SDS-PAGE results for Ni-NTA purification for gp15 and gp16. Abbreviations: Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (C) Chromatograms and SDS-PAGE results for gel filtration purified gp15 and gp16 on a calibrated analytical Superdex200 10/300 column. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection protein gp14 (A) SDS-PAGE results for sonicated, NLS detergent extracted, and Ni-NTA purified samples of gp14. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation, US = Ultracentrifuged Supernatant sample, UP = Ultracentrifuged Pellet sample, Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (B) Chromatogram and SDS-PAGE results for gel filtration purified gp14 solubilized in NLS (extraction) detergent. (C) Chromatogram and SDS-PAGE results for gp14 detergent exchange from NLS to DDM detergent using an anion exchange column. (D) Chromatogram and SDS-PAGE results for gel filtration purified gp15 solubilized in DDM (mild) detergent. Only relevant lanes from the same gel are shown in (B–D). Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Extraction, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: In vitro assembly of the T7 periplasmic tunnel from purified gp15 and gp16 (A) Diagram of complex components incubated in a 3:1 molar ratio on ice for 60 min (B) Chromatogram and SDS-PAGE analysis of gel filtrated gp15:gp16 sample. Magenta arrow represents gp15 expected molecular weight at 87 kDa. Green arrow represents gp16 expected molecular weight at 146 kDa. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: In Vitro, Purification, Incubation, SDS Page, Molecular Weight, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expected results for negative stain TEM and cryo-EM screening (A and B) Representative micrographs of (A) negatively stained and (B) vitrified gp15:gp16 complex. On the right-hand side are 2D-class averages. Scale bars, 100 nm. Reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Staining, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet:

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Virus, Recombinant, Electron Microscopy, Filtration, Plasmid Preparation, Software, Chromatography, Electrophoresis, Microscopy

Journal: Scientific Reports

Article Title: A fixation method for the optimisation of western blotting

doi: 10.1038/s41598-019-43039-3

Figure Lengend Snippet: Fixation-dependent differences in lectin staining intensities when using PVDF and nitrocellulose membranes. Pooled human serum proteins (3 μg) were separated on 10% SDS-PAGE, and the proteins were transferred onto PVDF and nitrocellulose membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments and followed by staining with lectins (LCA and SNA). Lane i, CBB staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, sample heating at 100 °C; lane v, organic solvent (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) treatments at room temperature; lane vi, organic solvent treatments followed by sample heating at 100 °C. All treatments were applied for 30 min. Left, WB pattern; right, quantitative analysis (n = 3 individual experiments). The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. ** Significantly different p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).

Article Snippet: Biotinylated lectins, LCA, SNA, PHA-E, PHA-L, and AAL (Supplementary Table ), were purchased from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: Staining, SDS Page, Software

Journal: Nature Structural & Molecular Biology

Article Title: Cryo-EM structure of the human CST–Polα/primase complex in a recruitment state

doi: 10.1038/s41594-022-00766-y

Figure Lengend Snippet: a , Zoomed-in view of the interaction of POLA1 CTD (cartoon) with CTC1 (cartoon and surface) superposed with POLA1 CTD from the apo Polα/primase structure (PDB ID: 5EXR ) . The CTC1-recognition loop (CRL, 1400–1424 aa) is highlighted in cyan in the cartoon representation. The corresponding cleft in CTC1 is indicated with a cyan arc. b , Comparison of POLA1 CTD models generated with AlphaFold 2 (refs. , ) from several species alongside domain comparisons of corresponding CTC1 orthologs. The metazoan CTC1 N-terminal expansion is indicated, and the binding pocket is represented by a cyan arc as in a or a dashed fuchsia arc in the non-metazoan species. c , Microscale thermophoresis measuring binding of POLA1 CTD proteins to RED-tris-NTA-labeled His 6 -MBP-CTC1–STN1–TEN1. Error bars represent s.e.m. for each data point, calculated from three independent thermophoresis measurements (Extended Data Fig. ). d , e , Zoom-in of binding events 1 and 2 from c , respectively. K D values were calculated (N.D., not determined) with the MO Affinity Analysis (Nanotemper) software (split into two events for the CRL WT and generated with all data for the CRL GGSGGS and CRL S. pombe mutants). F norm , normalized fluorescence.

Article Snippet: Gels are representative of three independent protein preparations. c , SEC-elution profiles of the POLA1 CTD constructs (estimated MW ~20 kDa) superposed with gel-filtration standards (BioRad). d , Models generated with AlphaFold 2 , of the human POLA1 CTD constructs encoding CRL WT (cyan), CRL GGSGGS (red), or CRL S. pombe (fuchsia), demonstrating the accommodation of shortened linkers. e , Independent microscale thermophoresis experiments with POLA1 CTD constructs and RED-tris-NTA-labeled His 6 -MBP-CTC1–STN1–TEN1.

Techniques: Generated, Binding Assay, Microscale Thermophoresis, Labeling, Software, Fluorescence

Journal: Nature Structural & Molecular Biology

Article Title: Cryo-EM structure of the human CST–Polα/primase complex in a recruitment state

doi: 10.1038/s41594-022-00766-y

Figure Lengend Snippet: a , Coomassie blue-stained SDS-PAGE gels (4–12% (w/v) Bis-Tris, run in MES-SDS; Invitrogen) showing proteins used for MST analyses. POLA1 CTD constructs run as heterogeneously sized smears on SDS-PAGE. Gels are representative of three independent protein preparations. b , Native PAGE (4–16% Bis-Tris; Invitrogen) gel showing POLA1 CTD constructs and a 1 μg BSA loading control next to a NativeMark unstained protein standard. Gels are representative of three independent protein preparations. c , SEC-elution profiles of the POLA1 CTD constructs (estimated MW ~20 kDa) superposed with gel-filtration standards (BioRad). d , Models generated with AlphaFold 2 , of the human POLA1 CTD constructs encoding CRL WT (cyan), CRL GGSGGS (red), or CRL S. pombe (fuchsia), demonstrating the accommodation of shortened linkers. e , Independent microscale thermophoresis experiments with POLA1 CTD constructs and RED-tris-NTA-labeled His 6 -MBP-CTC1–STN1–TEN1. Dissociation constant (K D ) values were calculated with the MO Affinity Analysis (Nanotemper) software. Error bars represent the s.e.m. for each data point calculated from three capillary scans. The combined data are shown in Fig. .

Article Snippet: Gels are representative of three independent protein preparations. c , SEC-elution profiles of the POLA1 CTD constructs (estimated MW ~20 kDa) superposed with gel-filtration standards (BioRad). d , Models generated with AlphaFold 2 , of the human POLA1 CTD constructs encoding CRL WT (cyan), CRL GGSGGS (red), or CRL S. pombe (fuchsia), demonstrating the accommodation of shortened linkers. e , Independent microscale thermophoresis experiments with POLA1 CTD constructs and RED-tris-NTA-labeled His 6 -MBP-CTC1–STN1–TEN1.

Techniques: Staining, SDS Page, Construct, Clear Native PAGE, Filtration, Generated, Microscale Thermophoresis, Labeling, Software

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , S -acylation is mediated by ZDHHC loading of long-chain acyl-CoA derived from lipid biosynthesis followed by acyl transfer to a proximal Cys of a protein substrate and regeneration of apo-ZDHHC. The reversible cycle is closed by thioester hydrolysis by APTs. b , X-ray structure of human ZDHHC20 irreversibly inhibited by lipid mimic 2-bromopalmitate (PDB ID: 6BML ). Inset, sterically demanding residues in the ZDHHC20 lipid-binding pocket contact the acyl chain distal to the DHHC catalytic site. c , Steric complementation between a ZDHHC ‘hole’ mutant and an alkyne-tagged ‘bumped’ lipid substrate probe enables selective loading and tag transfer to ZDHHC substrates, bypassing endogenous (WT) ZDHHCs. Fluorescence visualization and chemical proteomics are enabled by bioorthogonal conjugation to multifunctional capture reagents.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Derivative Assay, Binding Assay, Mutagenesis, Fluorescence, Conjugation Assay

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Fatty acid probes containing an alkynyl click-handle (blue), varying chain length L = 16, 18 or 20 heavy atoms in the chain (carbons + nitrogen) and an R ‘bump’ group (red)—Ac, c Pr or Bz. b , Two-stage pairing strategy for a designed ZDHHC20 mutant optimizes probe chain length and then bump size to match the new binding cavity, with probe activation, selectivity over ZDHHC20 WT and transfer to a known ZDHHC20 substrate (IFITM3) optimized in parallel. c – f , Bump-hole loading analysis of C-terminal FLAG-tagged ZDHHC20 WT and mutants in HEK293T cells treated with 15 μM YnPal ( c , d ) or 18-Ac ( e , f ) for 4 h (D, catalytic-dead ZDHHC20(C156S); E, empty vector; n = 3 independent biological replicates average ± s.d.). g , Probe bump-size optimization by transfer assays with HA-IFITM3 and either WT ZDHHC20 (W) or ZDHHC20(Y181G) (M) co-expression in HEK293T cells ( n = 3 independent biological replicates average ± s.d.). h , Average loading and transfer activity relative to highest fluorescent/input ratio ( n = 3 independent biological replicates average ± s.d.). i , j , Enzyme kinetics for WT ZDHHC20 and ZDHHC20(Y181G) treated with Pal-CoA ( i ) or 18-Bz-CoA ( j ) using a KDH assay ( 3 ). Michaelis–Menten plots generated from average reaction rate (NADH generated μM min −1 , n = 3 independent experiments) ± s.d. versus lipid concentration (μM). d , f , h , The two-tailed unpaired t test of Prism 9.0 was used to determine P values and noted above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Mutagenesis, Binding Assay, Activation Assay, Plasmid Preparation, Expressing, Activity Assay, Generated, Concentration Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a-b ) Catalytically dead ZDHHC20 is appreciably labeled by YnPal at peripheral cysteine sites. FLAG-tagged WT and ZDHHC20[C156S] constructs were transfected in HEK293T cells and treated with the indicated concentration of YnPal for 4 h. After lysis and IP with anti-FLAG resin, samples were subjected to CuAAC with TAMRA azide and separated by SDS-PAGE. ZDHHC20 loading and input were measured by in-gel fluorescence and anti-ZDHHC20 immunoblot (n = 3 independent biological replicates). ( c-d ) Thioester dependence of ZDHHC20 labeling was demonstrated upon treatment of YnPal and C18-Bz treated samples with 0.8 M neutralized NH 2 OH following IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( e-f ) Time-course measuring 15 µM YnPal labeling of ZDHHC20 WT expressing HEK293T cells (n = 3 independent biological replicates). ( g-h ) Labeling activity of the indicated concentrations of YnPal in FLAG-tagged ZDHHC20[Y181G] and ZDHHC20[Y181G/C156S] expressing HEK293T cells (n = 3 independent biological replicates). The average (n = 3 independent biological replicates) loading ( b , d , f and h ) was reported as a percent of the maximal fluorescent: input ratios ± S.D. between treatments with and without hydroxylamine. ( i-l ) Probe chain-length was optimized against ZDHHC20[Y181G] using cell-based loading ( i-j ) and transfer ( k-l ) assays in HEK293T using ZDHHC20 WT (W) and ZDHHC20[Y181G] (M). ( i ) HEK293T cells were treated with 15 µM acetyl bumped probes of L = 16, 18 and 20 for 4 h and enzyme loading assessed by in-gel fluorescence following anti-FLAG IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( k ) HEK293T cells co-expressing ZDHHC20[Y181G] and HA-Ifitm3 were treated with 15 µM 18-Ac or 20-Ac for 4 h with loading and transfer of the probe assessed following by anti-FLAG/anti-HA IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( j-l ) The average (n = 3 independent biological replicates) loading and transfer activity were reported as a percent of the maximal fluorescent/input ratios ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and are note above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Construct, Transfection, Concentration Assay, Lysis, SDS Page, Fluorescence, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Wild-type (WT), Y181G (YG), C156S (CS) and Y181G/C156S (YGCS) FLAG-tagged ZDHHC20 constructs were transfected into HEK293T cells and purified by anti-FLAG agarose affinity chromatography. After enzyme elution with 3X FLAG-peptide, buffer was exchanged using 50 kDa M.W. cut-off protein concentrator tubes and sample concentration determined using a BSA standard curve. All samples were run on SDS-PAGE gels and protein visualized by Coomassie staining (n = 2 independent experiments). ( b ) An enzyme-coupled assay monitoring ZDHHC20 autoacylation was established using commercial α-ketoglutarate dehydrogenase enzyme (KDH) along with its substrates α-ketoglutarate (α-KG), thiamine pyrophosphate (TPP) and NAD+. Optimization of α-ketoglutarate dehydrogenase (KDH) ( c ) and WT ZDHHC20 ( d ) concentrations. Pal-CoA ( e ) and 18-Bz-CoA ( f ) KDH activities were determined in the absence of ZDHHC20, to establish background rates for each probe. ( g ) 18-Bz-CoA displayed significant background activity in the KDH assay without ZDHHC20. Reaction rates for ZDHHC20[C156S] ( h ) and ZDHHC20[Y181G, C156S] ( i ) treated with Pal-CoA or 18-Bz-CoA. Michaelis-Menten plots generated by plotting average (n = 3 independent experiments) reaction rates (NADH generated (µM)/min) ± S.D.) versus lipid concentration (µM) using Prism 9.0. For reactions with 18-Bz-CoA, the basal rates at all concentrations tested were subtracted from the corresponding total reaction rates.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Construct, Transfection, Purification, Affinity Chromatography, Concentration Assay, SDS Page, Staining, Activity Assay, Generated

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: FLAG-tagged ZDHHC20 WT and ZDHHC20[Y181G] expressing HEK293T cells were treated with the indicated concentration of 18-Bz ( a-b ) for 4 h in cell-based loading assays (n = 3 independent biological replicates). ( c-d ) FLAG-tagged ZDHHC20[Y181G] and HA-Ifitm3 expressing HEK293T cells were treated with 15 µM 18-Bz for the indicated time in cell-based loading and transfer assays (n = 3 independent biological replicates). ( e-f ) FLAG-tagged ZDHHC20 WT and ZDHHC20[Y181G] expressing HEK293T cells were treated with 15 µM 18-Bz for the indicated times (n = 3 independent biological replicates). Lysates were clicked with TAMRA azide then analyzed by in-gel fluorescence and SDS-PAGE; note YG-dependent labeling of substrate protein bands (*). Input was assessed by anti-ZDHHC20 (D20) immunoblot. The average (n = 3 independent biological replicates) loading ( b, d & f ) and transfer ( d ) were reported as a percent of the maximal fluorescent: input ratios ± S.D. ( g-h ) The effect of FBS concentration on ZDHHC20 loading and transfer. ( g ) FLAG-tagged wild-type (WT) or ZDHHC20[Y181G] (M) and HA-Iftim3 expressing HEK293T cells were treated with 15 µM YnPal or 18-Bz in the presence of 0.5 or 10% FBS for 4 h in cell-based transfer assays. ( h ) The average (n = 3 independent biological replicates) loading and transfer were reported as a percent of the maximal fluorescent: input ratio ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and noted above the relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Expressing, Concentration Assay, Fluorescence, SDS Page, Labeling, Western Blot, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Left: representative confocal microscopy images showing average signal of Z-stacks of HEK293T cells transiently co-expressing ZDHHC20 WT HA-tagged and Y181G mutant FLAG-tagged. Each image shows signal for HA (magenta), FLAG (green), p-cadherin as plasma membrane marker (yellow), nucleus (blue) and a composite image of all signals. Scale bar at the bottom right marks 20 mm, while the other white line highlights the region of interest (ROI) used for image analysis. Right: plot showing normalized fluorescence signal for each of the channels in the ROI (1 biological replicate). ( b ) As a , but with using Gm130 as Golgi marker (1 biological replicate).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Confocal Microscopy, Expressing, Mutagenesis, Membrane, Marker, Fluorescence

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Chemical proteomic OBH workflow for enrichment and identification of S -acyltransferase substrates and S -acylation sites by LC–MS/MS. b , Chemical proteomic analysis of ZDHHC20 substrates in HEK293T cells (15 µM 18-Bz, 8 h). Enrichment in ZDHHC20(Y181G) cells over WT ZDHHC20 reveals selective ZDHHC20 loading (red triangle), and significantly enriched substrates (green circles) selected for further validation (red circles), with site identification data (blue triangles; Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). c , d , LC–MS/MS spectrum corroborating reported sites of CD151 ( c ) S -acylation at Cys11 and Cys15 and of STX7 ( d ) S -acylation at Cys28 (see also Extended Data Fig. ). e , S -acylated proteome profiling using YnPal. HEK293T cells transiently transfected with WT ZDHHC20 or ZDHHC20(Y181G) were treated with 15 µM YnPal for 8 h before processing using the on-bead digestion workflow. Substrates highlighted in green had been identified using a chemical–genetic system (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). f , g , Validation of S -acylation for substrates at endogenous levels. HEK293T cells transiently transfected with WT ZDHHC20 (W) or ZDHHC20(Y181G) (M) were treated with 15 µM 18-Bz ( f ) or 15 µM YnPal ( g ) for 24 h. Lysates were clicked with biotin azide before enrichment on neutravidin magnetic beads. Representative immunoblots are shown for input and pull-down signals ( n = 2 independent biological replicates). h , Venn diagram of putative ZDHHC20 substrates identified in HEK293T, MDA-MB231 and PANC1 cells. i , Statistical overrepresentation analysis of putative ZDHHC20 substrate cellular compartment (Slim)-GO terms compared to the full human genome using the PANTHER classification system showing terms with >9 −log ( P value) from an FDR-adjusted two-tailed Fisher’s exact test.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Transfection, Magnetic Beads, Western Blot

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Full gel and western blots of all replicates corresponding to the chemical proteomics ZDHHC20 substrate identification (Fig. ) performed in HEK293T cells. A portion of the lysate was clicked with TAMRA azide for analysis by in-gel fluorescence. The bands present at ~35 kDa in ZDHHC20[Y181G] (M) lanes but absent in WT ZDHHC20 lanes indicate selective loading of 18-Bz on ZDHHC20[Y181G] over WT-ZDHHC20. Anti-FLAG WB indicates similar expression levels of WT construct compared to ZDHHC20[Y181G] construct. Vinculin is used as loading control (n = 4 independent biological replicates). ( b-e ) Chemical proteomics ZDHHC20 substrate detection with 18-Bz probe (15 µM) in ( b ) PANC1 cells and (C) MDA-MB-231 cells. Cells were transiently transfected with WT ZDHHC20 versus ZDHHC20[Y181G] (M) then clicked with biotin azide and enriched on neutravidin agarose for proteomic processing. Significantly enriched putative substrates (Student’s two tailed unpaired t-test S0 - 0.5, adjusted FDR - 0.01) are shown as green circles, hits with site identification data are shown in as blue triangles and other validated substrates are highlighted as red circles. 200 putative ZDHHC20 substrates are identified in (B) PANC1 cells and 50 putative substrates in ( c ) MDA-MB-231 cells. ( d-e ) Gel and western blots corresponding to the volcano plot in ( a-b ) where a portion of the lysate was clicked with TAMRA azide as described in B (n = 4 independent biological replicates). (f) Statistical over/underrepresentation analysis of putative ZDHHC20 substrate biological process GO-terms compared to a reference list containing reported S -acylated proteins (SwissPalm) using the PANTHER classification system showing terms with >1.5 -Log 10 (p-value) from an FDR adjusted Fisher’s exact two tailed test. (g-h) PTRH2 Site ID analysis and quantification. (g ) Validation of HA-PTRH2 S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants. Representative images (n = 3 independent biological replicates) for TAMRA signal are shown, as well as for HA and FLAG immunoblots for HA pull down and input. Calnexin was used as loading control. (h ) Bar plot showing the ratio of TAMRA fluorescence and HA pulldown signal of PTRH2 cysteine mutants as a percentage of WT PTRH2 ratio. The two tailed unpaired t-test statistical module of Prism 9.0 was used to calculate p-values and noted above relevant comparisons. (i) Profiling of Flp-in T-Rex substrates ZDHHC20 cell lines. The average (n = 3 independent biological replicates) Fold change of FLAG signal is reported as a percent of the maximal ratios ± S.D. The unpaired t-test statistical module of Prism 9.0 was used to determine p-values and noted above relevant comparisons. Related to main Fig. .

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Western Blot, Fluorescence, Expressing, Construct, Control, Transfection, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a-b ) Gels and corresponding volcano plots for HEK293T cells treated with 15 μM 20- c Pr for 8 h in the presence of ZDHHC15 WT or ZDHHC15[Y184G]. ( a ) Lysates were subjected to CuAAC with TAMRA azide to assess probe incorporation and expression levels of FLAG-tagged ZDHHC and the loading control vinculin. ( b ) Volcano plot showing enrichment of putative ZDHHC15 substrates by ZDHHC15[Y184G] (Student’s two tailed unpaired T-test, S 0 0.5, adjusted FDR 0.01, n = 4 independent biological replicates) of matched lysates processed by OBH workflow and analyzed by LC-MS/MS. The positive control ZDHHC15 (red triangle) shows enrichment and many sites of modification (blue triangle) were identified through our OBH workflow. ( c-d ) Gels and corresponding volcano plots for PANC1 cells treated as described in ( a-b ). ( e-f ) Gels and corresponding volcano plots for HEK293T cells treated with 15 μM 20- Bz for 8 h in the presence of ZDHHC7 WT or ZDHHC7[L57G]. ( e ) Lysates were subjected to CuAAC with TAMRA azide to assess probe incorporation and expression levels of FLAG-tagged ZDHHCs and the loading control vinculin. ( f ) Volcano plot showing enrichment of putative ZDHHC7 substrates by ZDHHC7[L57G] (Student’s two tailed unpaired T-test, S 0 0.5, adjusted FDR 0.05, n = 4 independent biological replicates) of matched lysates processed by OBH workflow and analyzed by LC-MS/MS. ( g-h ) Overlap among ZDHHC substrate profiles for ZDHHC7, ZDHHC15, and ZDHHC20 (Student’s two tailed unpaired t-test, S 0 0.5, adjusted FDR 0.05, n = 4 independent biological replicates). ( g ) Volcano plot of ZDHHC20 OBH shown in Fig. , with unique putative substrates; putative substrates shared with ZDHHC7 and/or ZDHHC15 highlighted. ( h ) Volcano plot of ZDHHC7 OBH shown in Supplementary Fig. with unique putative substrates; putative substrates shared with ZDHHC15 and/or ZDHHC20 highlighted.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Expressing, Control, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Positive Control, Modification

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , b , ZDHHC20(Y181G) retains exquisite selectivity for specific cysteines on substrates IFITM3 ( a ) and PI4K2A ( b ; n = 3 independent biological replicates average ± s.d.), matching previously reported labeling, with the 18-Bz bumped probe. c , Validation of HA-STX7 S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants (C28A) and (C239A). Representative images ( n = 3 independent biological replicates average ± s.d.) for TAMRA signal are shown, as well as for HA and FLAG immunoblots for HA pull down and input. Calnexin was used as loading control. d , e , Validation of HA-VAMP3 and HA-BCAP31 site S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants, VAMP3(C76A) and BCAP31(C23A). d , Cell-based transfer assays were performed without FLAG-ZDHHC20 and HA-VAMP3 enrichment, but rather with direct labeling of cell lysates by TAMRA-azide click followed by SDS–PAGE and anti-HA, anti-FLAG and anti-vinculin immunoblot analysis. e , FLAG-ZDHHC20 and HA-BCAP31 constructs were enriched before TAMRA-azide click labeling. f , Confirmation of trans -auto- S -acylation in peripheral cysteines on a catalytically dead C-HA-ZDHHC20(C156S) (D) by a mutant C-FLAG-ZDHHC20(Y181G) (M) with 15 μM 18-Bz. Catalytically dead C-FLAG-ZDHHC20(Y181G) (DM) did not transfer the probe to D. Cells transfected with an empty vector (E) were used as negative control. HA- and FLAG-tagged ZDHHC20 constructs were transiently cotransfected into HEK293T cells and treated with 15 μM 18-Bz for 4 h. After cell lysis, constructs were separately enriched on anti-HA and anti-FLAG resins, clicked with TAMRA-azide and separated by SDS–PAGE. Loading and input were visualized by in-gel fluorescence and immunoblot, respectively. The average ( n = 3 independent biological replicates) loading and transfer activity were reported as a percent of the maximal fluorescent:input ratios ± s.d. a , c , f , The two-tailed unpaired t test of Prism 9.0 was used to determine P values and noted above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Western Blot, Control, SDS Page, Construct, Mutagenesis, Transfection, Plasmid Preparation, Negative Control, Lysis, Fluorescence, Activity Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Profile of WT ZDHHC20 (W) or ZDHHC20(Y181G) Flp-In 293 T-REx cell lines treated with 18-Bz (15 µM, 24 h). Lysates were clicked with TAMRA azide and then analyzed by in-gel fluorescence and SDS–PAGE. Note that the asterisk represents YG-dependent labeling of substrate protein bands. b , Comparison of protein expression levels between doxycycline induction of Flp-In 293 T-REx cells and overexpression by transient expression in HEK293T cells. Representative immunoblots are shown for FLAG at high or low exposure, to probe for ZDHHC20 WT versus ZDHHC20(Y181G), and calnexin as loading control ( n = 3 independent biological replicates). c , In Flp-In 293 T-REx cells ZDHHC20(Y181G) retains exquisite selectivity for its substrate IFITM3 with the 18-Bz bumped probe, as seen in prior experiments. d , Chemical proteomic analysis of ZDHHC20 substrates in Flp-In 293 T-REx cells (15 µM 18-Bz, 24 h). Enrichment in T-REx ZDHHC20(Y181G) cells over T-REx WT ZDHHC20 reveals selective ZDHHC20 modification of substrates (green) (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). e , f , Validation of S -acylation for T-REx ZDHHC20(Y181G) substrates at endogenous levels. Flp-In 293 T-REx cells, WT ZDHHC20 (W) or ZDHHC20(Y181G) (M), induced with doxycycline for 24 h, were treated with 15 µM 18-Bz ( e ) or YnPal ( f ) for 24 h. Lysates were clicked with biotin azide before enrichment on neutravidin magnetic beads. Representative immunoblots are shown for input and pull-down signals ( n = 2 independent replicates).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Fluorescence, SDS Page, Labeling, Comparison, Expressing, Over Expression, Western Blot, Control, Modification, Two Tailed Test, Magnetic Beads

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Untreated (UT) or gRNA/CAS9 treated (pSpCas9(BB)-2A-Puro, PX459 plasmid) HEK293T cells were probed with anti-ZDHHC20 (D20) and –vinculin antibodies. Cells treated with gRNA1/CAS9 resulted in knockdown (KD); whereas cells treated with gRNA2/CAS9 yielded two ZDHHC20-knockout (D20-KO) clones: KO1 and KO2 (n = 2 independent biological replicates). ( b ) WT or KO2 HEK293T cells were transfected with HA-IFITM3 and empty vector or C-FLAG-tagged ZDHHC20. Cells were then treated with 15 mM YnPal for 4 h before being harvested and lysed. IFITM3 and D20 were enriched in one pot with a mix of anti-HA and –FLAG resins before being treated with TAMRA-azide and click reagents. Tagged proteins were eluted from beads with 1X Laemmli buffer and separated by SDS-PAGE. YnPal ZDHHC20-loading and transfer to IFITM3 and input were visualized by in-gel fluorescence and anti-HA and -FLAG immunoblot, respectively (n = 2 independent biological replicates). ( c ) The average (n = 3 independent biological replicates) loading and transfer activity was reported as a percent of the maximal D20 fluorescent: input ratio and as a percent of the WT IFITM3 (empty vector) fluorescent: input ratio ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and noted above relevant comparisons ( d-g ) WT HEK293T cells, two ZDHHC20 KO clones, and one partial knockdown (KD) clone were treated with 15 μM YnPal for 8 h. As a control for lipidation, HEK293T cells were treated with palmitic acid (Pal) and also taken through the experiment. Samples were then clicked with biotin-TAMRA-azide, 10% of which was analyzed by SDS-PAGE, in-gel fluorescence, and anti-tubulin western blot ( d ) (n = 3 independent biological replicates). The remainder was enriched on dimethylated neutravidin beads and digested for LC-MS/MS LFQ analysis. (E-G) Whilst a small number of proteins are identified as being significantly enriched/depleted (Student’s two tailed unpaired T-test S 0 – 0.1, adjusted FDR – 0.05), they are few in number and none are consistently found which correspond to our putative chemical genetic substrates found in HEK293T cells. ( f ) Analysis of YnPal treated cells against Pal shows a large number a potentially lipidated proteins have been identified, with many well validated S -acylation proteins identified, some of which have been highlighted in blue.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Plasmid Preparation, Knockdown, Knock-Out, Clone Assay, Transfection, SDS Page, Fluorescence, Western Blot, Activity Assay, Two Tailed Test, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Schematic representation of TurboID fusion proteins used for proximity labeling experiments. ( b-c ) Confirmation of the expression of each fusion protein by western blot after generation of ‘Jump-in’ cell lines using either anti-V5 antibody ( b ) (n = 2 independent biological replicates) or an anti-GFP antibody ( c ) (n = 2 independent biological replicates). The labeling efficiency of the TurboID biotin ligase was confirmed by the addition of 500 μM biotin for the indicated times. Only those cells expressing the ligase show an increase in the biotinylation of proteins, as determined by Streptavidin conjugated HRP, compared to the UT HEK293T cells, and also in a time dependent manner. ( d ) Volcano plot showing the enrichment of proteins when comparing the C-terminally tagged ZDHHC20 with the N-terminally tagged construct (Student’s two tailed unpaired t-test S 0 – 0.1, adjusted FDR 0.01). There does appear to be a preference for either the N- or C- terminus for some interactors, none of these correspond to our chemical genetic hits. ( e ) Volcano plot showing the enrichment of proteins when comparing the N-terminally tagged ZDHHC20 with the Turbo GFP construct (Student’s two tailed unpaired t-test S 0 – 0.1, adjusted FDR 0.01). ( f ) TurboID-based proximity-labeing enabled detection of ZDHHC20 (D20) interactors. Volcano plot showing the mean log 2 difference in protein group intensities between N-TurboID-ZDHHC20 and TurboID-GFP clones (Student’s two tailed unpaired T-test S 0 – 0.1, adjusted FDR 0.01).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Expressing, Western Blot, Construct, Two Tailed Test, Clone Assay

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Structure-guided ZDHHC engineering exemplified for ZDHHC7 (see also Extended Data Figs. and ). ZDHHC7 homology model (yellow/orange) overlayed on experimental ZDHHC20 structure (dark green) identifies a potential hole-generating amino acid (Leu57) on an adjacent helix in the vicinity of ZDHHC20 Tyr181; lipid density (blue mesh), and length/size probe analysis identifies a mutant/probe pair (ZDHHC7(L57G)/20-Bz) with optimal activity and selectivity over WT ZDHHC7. b , Bump-hole analysis of N-FLAG-tagged WT ZDHHCs or mutant ZDHHCs ZDHHC3(I182G) (D3), ZDHHC7(L57G) (D7), ZDHHC11(M181A) (D11) and ZDHHC15(Y184G) (D15) in HEK293T cell-based loading assays using 15 µM corresponding optimized probe. c , Average ( n = 3 independent biological replicates) loading reported as a percent of maximal fluorescent:input ratio ± s.d. P values determined by Prism 9.0 two-tailed unpaired t test statistical module and noted above relevant comparisons. d , ZDHHC15 substrate discovery in HEK293T cells treated with 15 µM 20- c Pr in HEK293T cells using the OBH workflow. In total, 107 chemical–genetic ZDHHC15 substrates were identified (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates). Substrates unique or in common with parallel analyses for DHHC7 and DHHC20 in HEK293T cells are highlighted (Extended Data Fig. ). e , Overlap of chemical–genetic ZDHHC substrates identified in HEK293T cells. Of 301 total substrates, only 87 are shared by 2 or more family members, suggesting distinct substrate pools for each ZDHHC.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Mutagenesis, Activity Assay, Two Tailed Test

Journal: PLoS Genetics

Article Title: The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation

doi: 10.1371/journal.pgen.1008331

Figure Lengend Snippet: A . Apoptosis signal in HaEpi cells after DMSO, 20E (5 μM), dsGFP +20E (5 μM), dsDopEcR+ 20E (5 μM) and DA (10 μM) +20E (5 μM) treatment by using the NucView caspase-3 activity assay kit. a . The ratio of apoptotic cells (green) to the total cells (blue) in the field view was obtained. B . Proliferation signal in HaEpi cells after DMSO, 20E (5 μM), dsGFP +20E (5 μM), dsDopEcR+ 20E (5 μM) and DA (10 μM) +20E (5 μM) treatment by using the 5-ethynyl-2′-deoxyuridine (EdU) kit (Ribobio, Guangzhou, China). b . The ratio of proliferation cells (green) to the total cells (blue) in the field view was obtained. DAPI stained the nucleus (blue). Statistical analysis using the data from 100 × 3 cells. The yellow bars represent 100 μM. C . Western blot analysis of 20E (5 μM) or DA (10 μM) induced proteins phosphorylation for 30 min. a. b . and c . AKT-RFP-His, AKT -RFP-His, and CDK10-RFP-His. 7.5% SDS-PAGE gel. Error bars show the mean ± SD of three times’ biological repetition. ImageJ software was used to transform the image data. Significant differences were calculated by Student’s t test (* p < 0.05; ** p < 0.01) or one-way analysis of variance (ANOVA, p < 0.05).

Article Snippet: The NucView 488 caspase-3 assay kit (NO. 30029 Biotium, Hayward, USA) was used to detect the activity of caspase-3 in the HaEpi cells, according to the manufacturer’s instructions.

Techniques: Caspase-3 Activity Assay, Staining, Western Blot, SDS Page, Software

Journal: Nucleic Acids Research

Article Title: RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts

doi: 10.1093/nar/gkab479

Figure Lengend Snippet: Depletion of AtRNH1C suppresses the assembly of RecA1 filaments. ( A ) Comparison of the structures of RecA1 in reca1 , reca1 / atrnh1c, why1 / 3 / atrnh1c and CM - AtRNH1C -3 xFLAG / reca1 backgrounds (left panel). For quantification, the lengths of at least 100 fluorescence signals were measured (right panel). Data are mean ± SD. Statistical testing was done using t- test. **** P < 0.0001; n.s, not significant. Bar = 5 μm. ( B ) ChIP analysis showed the enrichment of RecA1-GFP at rDNA regions. Col-0 was included as negative ChIP control. Four loci in rDNA regions were examined by qPCR. The mitochondrial gene Cox2 was used as negative control locus. The IP/Input ratios were normalized to Col-0. Three biological replicates were performed and indicated as open circles. Graphs represent mean ± SD. Statistical testing was done using multiple t- test. ** P < 0.01; *** P < 0.001. ( C ) Slot-blot assay of the overall R-loops in chloroplast DNA of RecA1 - GFP / reca1 and OE - AtRNH1C - 3xFLAG / RecA1 - GFP / reca1 treated with or without CIP. RNase H treated DNA was used as control. RNA:DNA hybrids were detected using S9.6 antibody (left panel). SYBR Gold staining indicated DNA loading (right panel). ( D ) The structure of RecA1 in seedlings treated with or without CIP (left panel). For quantification, the length of about 100 fluorescence signals were measured (right panel). Data are mean ± SD. Statistical testing was done using t- test. **** P < 0.0001; n.s, not significant. Bar = 5 μm. ( E ) PFGE detected the cpDNA damage in seedlings treated with or without CIP. The left panel shows the image of the gel after staining with ethidium bromide. The right panel is blot hybridization of the probe 55677–56181 (a 505-bp rbcL gene fragment). Arrowheads indicate the bands representing monomer and dimer cpDNA molecule.

Article Snippet: Chloroplast DNA was treated with 1 U of RNase III (New England Biolabs) per 5 μg of DNA at 37°C for 2 h, then purified with HiPure Gel Pure DNA Mini Kit (Magen).

Techniques: Fluorescence, Negative Control, Slot Blot Assay, Staining, Hybridization

Journal: Nucleic Acids Research

Article Title: RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts

doi: 10.1093/nar/gkab479

Figure Lengend Snippet: AtRNH1C and WHY1/3 co-localize at the same sites in the chloroplast genome. ( A , B ) WHY1 coimmunoprecipitation with AtRNH1C. The three-week-old seedlings of OE - WHY1 - GFP / OE - AtRNH1C - HA were used for immunoprecipitation with anti-GFP (A) and anti-HA (B) beads. Then the immunoprecipitated protein was detected with anti-HA and anti-GFP antibodies. ( C– E ) WHY1 co-immunoprecipitation with AtRNH1C depends on DNA/RNA. After immunoprecipitation with anti-HA beads as performed in (B), anti-GFP antibody was used to detect WHY1 protein. RT-PCR and digestion control were performed by amplifying an rbcL fragment from cDNA reverse transcribed from RNA or DNA isolated from beads with or without nuclease treatment, and primers are listed in . ( F ) Co-IP assay with CM - AtRNH1C -3 xFLAG complemental line. The anti-FLAG beads were used for immunoprecipitation. Col-0 was included to indicate the specificity of anti-FLAG beads. AtRNH1C-3xFLAG and WHY1/3 were detected with anti-FLAG and anti-WHY1/3 antibodies. RNase H treatment was performed to examine the dependency on RNA:DNA hybrids. ( G ) Fluorescence micrographs showing the co-localization of WHY1 with AtRNH1C. Plasmids encoding fusion proteins WHY1-mCherry and AtRNH1C-eGFP were transiently expressed in Arabidopsis protoplasts. Red indicates chloroplast autofluorescence; magenta indicates mCherry fluorescence; green indicates eGFP signals. ( H ) Immunofluorescence analysis of the co-localization of WHY1/3 and AtRNH1C. The chloroplasts from OE-AtRNH1C-3xFLAG plants were used for detection. Red indicates chloroplast autofluorescence; yellow indicates WHY1/3 proteins detected by anti-WHY1/3 antibody; green indicates AtRNH1C-3xFLAG proteins detected by anti-FLAG antibody; blue indicates the nucleoid detected by DAPI staining. ( I ) ChIP-reChIP analysis of AtRNH1C and WHY1/3 co-occupation at chloroplast genome. The chloroplast chromatin of OE-AtRNH1C - 3xFLAG was first immunoprecipitated by anti-WHY1/3 polyclonal antibodies and then by anti-FLAG antibodies. Protein G was used as a negative control. Four loci indicated in Figure were used for qPCR. The left panel shows mean ± SD ( n = 3 biologically independent samples, indicated as open circles). The right panel shows one representative gel image of sonicated cpDNA. M, Marker; S, Sonicated.

Article Snippet: Chloroplast DNA was treated with 1 U of RNase III (New England Biolabs) per 5 μg of DNA at 37°C for 2 h, then purified with HiPure Gel Pure DNA Mini Kit (Magen).

Techniques: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Isolation, Co-Immunoprecipitation Assay, Fluorescence, Immunofluorescence, Staining, Negative Control, Sonication, Marker

Journal: Nucleic Acids Research

Article Title: RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts

doi: 10.1093/nar/gkab479

Figure Lengend Snippet: WHY1/3 works in concert with AtRNH1C to regulate R-loops. ( A ) ChIP analysis showed the enrichment of WHY1/3 at rDNA regions. The why1 / 3 mutant line was included as negative ChIP control. The IP/Input ratios were normalized to why1 / 3 . ( B ) DRIP was performed with cpDNA isolated from Col-0, why1 / 3 , reca1 , why1 / 3 / reca1 , atrnh1c , why1 / 3 / atrnh1c and reca1 / atrnh1c . cpDNA of Col-0 pretreated with RNase H was included as negative control. The DRIP/Input ratios were normalized to Col-0. ( C ) Immunoblots showing the level of the PEP complex (BN-PAGE) as well as the total amount of RpoB (SDS-PAGE) in Col-0, why1 / 3 , reca1 , atrnh1c , why1 / 3 / reca1 , why1 / 3 / atrnh1c and reca1 / atrnh1c . Actin was used as loading control. The relative protein level of RpoB was quantified with ImageJ and indicated at the bottom. ( D ) ChIP analysis showed the enrichment of RpoB at rDNA regions. The Col-0 incubated with Protein G was used as negative ChIP control. ( E ) ChIP analysis showed the enrichment of AtRNH1C-3xFLAG at rDNA regions. The Col-0 was used as a negative control. The primers used in Figure were adapted for qPCR in these experiments. The IP/Input ratios were normalized to negative ChIP control. Three biological replicates were performed. Graphs represent mean ± SD. The significance was calculated by t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Chloroplast DNA was treated with 1 U of RNase III (New England Biolabs) per 5 μg of DNA at 37°C for 2 h, then purified with HiPure Gel Pure DNA Mini Kit (Magen).

Techniques: Mutagenesis, Isolation, Negative Control, Western Blot, SDS Page, Incubation