Journal: Heliyon

Article Title: A screening method for plastic-degrading fungi

doi: 10.1016/j.heliyon.2024.e31130

Figure Lengend Snippet: Raman spectroscopy reveals structural changes of plastics due to overgrowth of (A) Rhodotorula dairenensis EXF-13500 on PP, (B) Cladosporium sp. EXF-13502 on PA and (C) Rhodotorula sp. EXF-10630 on LDPE foil. The plastics polymers are low-density polyethylene (LDPE), and polyamide (PA), polyethylene terephthalate (PET), polypropylene (PP).

Article Snippet: Using a confocal Raman spectrometer (Integra Spectra I, NT-MDT Co., Russia), a blue wavelength laser with 488 nm was focused on the plastic sample surface at positions with and without fungi, in Raman shift range from 200 to 3000 cm −1 .

Techniques: Raman Spectroscopy

Journal: Nanomaterials

Article Title: Oriented Carbon Nanostructures from Plasma Reformed Resorcinol-Formaldehyde Polymer Gels for Gas Sensor Applications

doi: 10.3390/nano10091704

Figure Lengend Snippet: ( a ) XRD spectra and ( b ) Raman spectra of CNs obtained after the time-dependent plasma surface treatment on RF gel at 250 W and flow rate 100:50 sccm.

Article Snippet: Raman spectra were recorded at various points of the samples to study the structural changes of the nanostructures after plasma treatment using NTEGRA confocal Raman spectrometer (NT-MDT SI, Moscow, Russia) at an excitation wavelength of 633 nm with an incident power ~3 mW at a spot size of 50 μm.

Techniques: Clinical Proteomics

Journal: Nanomaterials

Article Title: Oriented Carbon Nanostructures from Plasma Reformed Resorcinol-Formaldehyde Polymer Gels for Gas Sensor Applications

doi: 10.3390/nano10091704

Figure Lengend Snippet: Gas sensor properties of OCNs prepared from RF gel: ( a ) sensor response to ethanol vapor at different heating temperatures, ( b ) repeatability of sensor response towards ethanol vapor; ( c ) sensor response towards the ethanol concentration as a function of heating temperature; ( d ) response and recovery time of OCN-based sensors towards ethanol at different heating temperature; and ( e ) ex-situ Raman spectra recorded on OCN sensor during the recovery of ethanol after sensor response at an acquisition time of 4 s with an interval of 6 s.

Article Snippet: Raman spectra were recorded at various points of the samples to study the structural changes of the nanostructures after plasma treatment using NTEGRA confocal Raman spectrometer (NT-MDT SI, Moscow, Russia) at an excitation wavelength of 633 nm with an incident power ~3 mW at a spot size of 50 μm.

Techniques: Concentration Assay, Ex Situ

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: A) Overview of recovery conditions. Following AY9944 sterol depletion, HEK293T hCLTA-Tq2 EN cells were exposed to cholesterol-loaded MβCD (MβCD-Chol, and chased in LPDS media) [Top] , supplemented continuously with lipoprotein-rich 15% FBS in the presence of AY9944 [Middle] , or incubated continuously in 7.5% LPDS media in the absence of AY9944 to allow endogenous cholesterol synthesis [Bottom] . B) Mid-plane live-cell confocal microscopy of HEK293T hCLTA-Tq2 EN following addition of transferrin-conjugated to AF-555 (Tfn-555) during recovery as described in (A). Representative images from different fields of view shown. Tfn and clathrin distributions in normal and arrested CME [Inset] . Scale bar = 10 μm. C) Corresponding cellular sterol profiles quantified by GC/MS (Mean ± SD). N = 3 biological replicates from independent experiments. Dotted and dashed lines represent total sterol abundance of untreated FBS and LPDS cultured cells, respectively.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Incubation, Confocal Microscopy, Gas Chromatography-Mass Spectrometry, Cell Culture

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: The ability of structurally diverse sterols to rescue CME inhibition due to sterol depletion by AY9944 treatment was evaluated by direct delivery of sterols to the PM via MβCD carrier. A) Summary of physical properties and phase separation behavior of sterols. Topological Polar Surface Area (TPSA) computed surface sum over all polar atoms. Ordered lipid ( L o ) domain (raft) stabilizing (+) or disrupting (−) sterols indicated relative to cholesterol (++). Refer to Table S2 for additional details. B) Sterol profiles of AY9944 treated HEK293T cells following 1h incubation with sterol loaded MβCD (Mean ± SD). N = 4 independent biological replicates from two MβCD-sterol preparations. C) Tfn uptake relative to controls cultured in 7.5% LPDS for 48 h (Mean ± SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA (F(12, 51) = 11.23, p < 0.0001) and Dunnett’s test versus LPDS control (N = 5 biological replicates from 3 independent experiments, ~1,500 cells per replicate). Cholesterol, desmosterol, 7DHC, and lathosterol support formation of L o domains in model membranes as designated. D) Representative confocal images taken mid-plane following 30 min incubation with AF-555 conjugated transferrin (Tfn). Scale bar = 20 μm. (c), Commercially available, pre-loaded MβCD-Chol.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Inhibition, Incubation, Cell Culture, Control

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: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a – d ) Sensitivity of WT and Rnf168 −/− MEFs to ICRF-193 ( a , b ) or etoposide ( c , d ) was determined using clonogenic assays. ( e , f ) Sensitivity of the human breast cancer cell lines T47D and MDA-MB-231 to etoposide was determined using clonogenic assays. ( a , c , e ) Data are presented as the mean±s.e.m. ( a , c , n >4; e , n =4). * P <0.05 for Rnf168 −/− MEFs compared with WT MEFs and T47D and MDA-MB-231 cells knocked down for RNF168 (sh.RNF168) compared with their respective controls (Sh.Ctr: ShRNA control). ( b , d ) Representative pictures of dishes showing surviving WT and Rnf168 −/− colonies post ICRF-193 ( b ) or etoposide ( d ) treatment. ( f ) Representative pictures of dishes showing surviving colonies of etoposide treated T47D and MDA-MB-231 cells.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: shRNA, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: PLK3 localizes at plasma membrane and Golgi apparatus. ( A ) RPE cells were transfected with non-targeting control siRNA or two siRNAs targeting PLK3. After 48 h, whole cell lysates were separated by SDS-PAGE and probed with indicated antibodies. Numbers indicate mobility in kDa. Position of PLK3 is indicated by arrowheads. Importin was used as loading control. ( B ) RPE cells were transfected with non-targeting control siRNA or siRNA to PLK3 or PLK1. Whole cell lysates were probed with rabbit monoclonal PLK3 antibody (Cell Signaling). Protein 143−3− was used as loading control. The position of PLK3 is indicated by arrowheads. ( C ) HEK293 cells stably transfected with EGFP-PLK3 grown on coverslips were fixed and probed with antibodies against GM130 (Golgi marker) and γTubulin (centrosome marker) and imaged by confocal microscopy. The arrowhead indicates the position of the centrosome. A representative image is shown. Bar indicates 5 μm. ( D ) RPE cells transfected with EGFP-PLK3 were fixed and probed with antibodies against GM130 and γTubulin. The arrowhead indicates the position of the centrosome. A representative image is shown. Bar indicates 5 μm. ( E ) HEK293 cells expressing EGFP2−93 were fixed and probed with indicated antibodies. Bar indicates 5 μm. ( F ) Cell lysates from HEK293 and HEK293 transfected with EGFP-PLK3 or EGFP were probed with indicated antibodies by immunoblotting. Note that only PLK3 antibodies from Cell Signaling and St. Johns recognize overexpressed EGFP-PLK3. Antibody from St. Johns also shows a large number of cross-reacting bands. ( G ) HEK293 cells stably transfected with EGFP-PLK3 were fixed 2 h after exposure to mock or to UVC and imaged using confocal microscopy. Bar indicates 5 μm. ( H ) HEK293 cells stably transfected with EGFP-PLK3 were treated with leptomycin and proteasomal inhibitor MG132 for 3 h, fixed and imaged using confocal microscopy. ( I ) Cytosolic, nuclear soluble and chromatin fractions of RPE cells were probed with indicated antibodies. Tubulin is a marker of cytosol, CHK2 of the nuclear soluble fraction and histone H2AX of chromatin. Asterisk indicates non-specific band of PLK3 antibody.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Clinical Proteomics, Membrane, Transfection, Control, SDS Page, Stable Transfection, Marker, Confocal Microscopy, Expressing, Western Blot

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Distribution of EGFP-PLK3 during the cell cycle. ( A ) Cells were synchronized at G1/S transition with thymidine block, released to the fresh media containing nocodazole and collected in 2 h intervals. Last sample was collected by mitotic shake-off. Histone H3-pS10 is a marker of mitotic cells. Asterisk indicates non-specific reactivity. ( B ) HEK293 cells stably expressing EGFP-PLK3 were fixed, stained with an antibody against γ-tubulin, and analyzed by confocal microscopy. Maximal projections of representative cells in various phases of the cell cycle are shown. Bar indicates 10 μm.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Blocking Assay, Marker, Stable Transfection, Expressing, Staining, Confocal Microscopy

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Generation of RPE-PLK3 knock-out cells. ( A ) Schematic diagram of CRISPR/Cas9-mediated targeting of human PLK3 and of domain organization of PLK3 protein. Black arrowhead indicates the position of the critical ATP-binding Lysine 91 in the catalytic domain. Empty arrowhead indicates the position of the epitope of the PLK3 antibody from Cell Signaling. Fragments of the genomic DNA from selected clones were PCR amplified and analyzed by sequencing. ( B ) Parental RPE and selected clones of RPE-PLK3-KO cells were analyzed by immunoblotting using rabbit monoclonal PLK3 antibody (Cell Signalling). ( C ) Parental RPE and two independent clones of RP-PLK3-KO cells were probed with indicated antibodies. The position of PLK3 is indicated by an arrowhead. Please note the missing band corresponding to PLK3 in staining with antibody from St Johns laboratory and high level of cross-reactivity with other proteins.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Knock-Out, CRISPR, Binding Assay, Clone Assay, Amplification, Sequencing, Western Blot, Staining

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Loss of PLK3 does not affect cell response to various forms of stress. ( A ) RPE or RPE-PLK3-KO cells were exposed or not to UVC, collected after 30 min and whole cell lysates were analyzed by immunoblotting ( n = 3). Antibody against γH2AX was used as a marker of induced DNA damage and TFIIH was used as a loading control. ( B ) RPE or RPE-PLK3-KO cells were incubated with isotonic, hypertonic or hypotonic media for 20 min and whole cell lysates were analyzed by immunoblotting. Antibody against active the form of p38-pT180/pY182 was used as a marker of cellular stress and TFIIH as loading control ( n = 3). ( C ) RPE cells transfected with control or two various PLK3 siRNAs were treated as in A and analyzed by immunoblotting ( n = 2). ( D ) RPE cells transfected with control or two various PLK3 siRNAs were treated as in B and analyzed by immunoblotting ( n = 2). ( E ) Validation of PLK3 siRNA using RT-qPCR. PLK3 mRNA level was normalized to ATP5B. ( F ) RPE or RPE-PLK3-KO cells were treated or not with indicated doses of CoCl2 for 24 h and whole cell lysates were analyzed by immunoblotting ( n = 3). ( G ) RPE and HeLa cells transfected with control or two various PLK3 siRNAs were treated or not with CoCl2 for 24 h and analyzed by immunoblotting ( n = 2 for each cell type). ( H ) RPE or RPE-PLK3-KO cells were exposed or not to 3 Gy of ionizing radiation and fixed after 1 or 8 h. Number of γH2AX positive foci per nucleus was determined by ScanR microscopy. Lines indicate median. ( I ) RPE or RPE-PLK3-KO cells were treated as in H and whole cell lysates were analyzed by immunoblotting ( n = 2). ( J ) RPE cells transfected with control or two various PLK3 siRNAs were treated as in H and whole cell lysates were analyzed by immunoblotting ( n = 2). ( K ) Cells were transfected with the wild-type PLK3 or kinase-dead K91R mutant; PLK3 was isolated by GFP Trap and incubated with casein in kinase buffer supplemented with radioactive ATP. Phosphorylation of casein was detected by autoradiography. ( L ) Cells were exposed to various forms of stress including treatment with CoCl2 (300 μM, 12 h), exposure to UV (10 J/m 2 ), etoposide (4 μM, 1 h), hypertonic media (480 μM mannitol) or left untreated. EGFP-PLK3 was isolated using GFP Trap and kinase assay was performed as in H. Induction of stress pathways was analyzed by immunoblotting of cell extracts. Arrowhead indicates identical position on the electrophoretic gel. ( M ) Quantification of the kinase assay in L. Signal was normalized to the non-treated control. Plotted is median +/− SD ( n = 3). Statistical significance was evaluated by two-tail t -test and significance was set to p < 0.05. Ns. stands for non-significant.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Western Blot, Marker, Control, Incubation, Transfection, Biomarker Discovery, Quantitative RT-PCR, Microscopy, Mutagenesis, Isolation, Phospho-proteomics, Autoradiography, Kinase Assay

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: PP6 phosphatase interacts with PLK3. ( A ) Lysates from HEK293 cells expressing EGFP or EGFP-PLK3 were incubated with GFP-Trap beads, washed three times and bound proteins were analyzed by mass spectrometry. Three biological replicates were performed. Numbers indicate average log difference between EGFP-PLK3 and EGFP. Listed are top 20 hits. ( B ) EGFP or EGFP-PLK3 was immunoprecipitated from stably transfected cells, and bound proteins were analysed by immunoblotting. Representative image is shown. ( C ) Lysates from HEK293 cells transfected with EGFP-PLK3, EGFP-PLK3-∆PBD or EGFP-PLK3-H590A-K592M (referred to as PLK3-HM) mutant were incubated with GFP-Trap and bound proteins were analysed by immunoblotting. ( D ) Cells expressing EGFP-PLK3 were transfected with control siRNA or siRNA to PP6C. After 48 h, cells were fixed, stained with antibodies against PP6C and γ-Tubulin and imaged using confocal microscopy. Arrowheads indicate the position of the centrosome. ( E ) Cells expressing EGFP-PLK3 were transfected with control siRNA or siRNA to PPP6R3. After 48 h, cells were fixed, stained with antibodies against PPP6R3 and γ-Tubulin, and imaged using confocal microscopy. Arrowheads indicate the position of the centrosome. ( F ) Validation of the knock-down efficiency in D and E. Cells transfected with control siRNA or siRNA to PPP6C, PPP6R1 or PPP6R3 were lysed and probed with indicated antibodies.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Expressing, Incubation, Mass Spectrometry, Immunoprecipitation, Stable Transfection, Transfection, Western Blot, Mutagenesis, Control, Staining, Confocal Microscopy, Biomarker Discovery, Knockdown

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: PP6 controls the phosphorylation level of PLK3. ( A ) Conservation of the T-loop of human PLK14−. Threonine residue corresponding to the T210 in PLK1 and to T219 in PLK3 is shown in bold. ( B ) EGFP-PLK3-WT and EGFP-PLK3-T219A was immunoprecipitated from transiently transfected cells using GFP-Trap and was probed with antibody against pT210-PLK1 or GFP. ( C ) EGFP-PLK3 was isolated from cells treated with mock or calyculin using GFP Trap. Isolated EGFP-PLK3 was incubated or not with lambda phosphatase for 20 min. All samples were probed with pT210 antibody. ( D ) HEK293-EGFP-PLK3 cells were transfected with PP6C and/or PPP6R1, and PLK3 was isolated using GFP-Trap. Level of PLK3 phosphorylation was evaluated by pT210 antibody. ( E ) HEK293-EGFP-PLK3 cells were transfected with negative control siRNA or siRNA to PP6C and grown for 48 h. Where indicated, cells were treated with calyculin 20 min prior to harvesting. PLK3 was isolated with GFP Trap. Level of PLK3 phosphorylation was evaluated by pT210 antibody. Kinase assay was performed as in F. ( F ) EGFP-PLK3-WT, EGFP-PLK3-T219A, EGFP-PLK3-T219D or a kinase dead EGFP-PLK3-K91R were purified from transiently transfected 293 cells using GFP Trap and incubated with casein and 32 P-γ-ATP for 20 min at 30 °C ( n = 2). Phosphorylation was detected by autoradiography and amount of precipitated PLK3 by staining with GFP antibody. Arrowhead indicates identical position of the casein on the electrophoretic gel. ( G ) EGFP or EGFP-PLK3-WT was purified from 293 cells transfected with mock, PP6C or PP6C with PPP6R1, and kinase assay was performed as in F. Phosphorylation of PLK3 was evaluated with pT210-PLK1 antibody and phosphorylation of the casein by autoradiography ( n = 2). Arrowhead indicates an identical position on the gel.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Residue, Immunoprecipitation, Transfection, Isolation, Incubation, Negative Control, Kinase Assay, Purification, Autoradiography, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: Increased SUMOylation of PML, STAT1 and UBC9 by IFNα. A, Relative changes in abundances of PML, STAT1 and UBC9 SUMOylation in response to IFNα, as determined by quantitative mass spectrometry. Means and standard deviations of three technical replicates are shown. B, Relative changes in PML, STAT1 and UBC9 protein expression in response to IFNα, as determined by Western blotting. C–E, PML, STAT1 and pSTAT1 SUMOylation as determined by Ni-NTA. C, HEK293-wt and HEK293-SUMO3m cells untreated or treated with the proteasome inhibitor for 4 h, were incubated with IFNα or IFNγ for 0.75 h, immunoblots of Ni-NTA-purified extracts were performed using anti-SUMO2/3 or anti-PML antibodies. D, HEK293-wt and HEK293-SUMO3m cells were untreated or treated with IFNα for 0.75 h. Inputs and immunoblots of Ni-NTA-purified extracts were performed using anti-STAT1 or anti-pSTAT1 antibodies. E, HEK293-wt and HEK293-SUMO3m cells untreated or treated with the proteasome inhibitor for 4 h, were incubated with IFNα for 0.75 h. Inputs (upper panel) and immunoblots of cytoplasmic and nuclear Ni-NTA-purified extracts (lower panel) were analyzed by Western blotting using anti-His, anti-H3 and anti-pSTAT1 antibodies. See also supplemental Fig. S3.

Article Snippet: Recombinant human IFNα2 was from Schering (USA), human IFNγ from Roussel Uclaf (Romainville, France), recombinant murine IFNα and IFNγ from R&D Systems (Minneapolis, MN).

Techniques: Mass Spectrometry, Expressing, Western Blot, Incubation, Purification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: PML is required for IFN-enhanced SUMOylation. A, B, wt MEFs and PML−/− MEFs were untreated or treated with murine IFNα for short (0.75 and 1 h) (A) or long (16 h) (B) periods and their extracts were analyzed by Western blotting for SUMO2/3, SUMO1, PML or Actin. C, D, PML−/− MEFs were transduced with HIV-1 derived lentiviral vectors expressing each human PML isoform (C) (PMLI, PMLII, PMLV, PMLVI or PMLVII), (D) (PMLIII or PMLIV). Two days post-transduction, PML expressing cells were untreated or treated with murine IFNα for 16 h and their extracts were analyzed by Western blotting for SUMO2/3, PML and Actin.

Article Snippet: Recombinant human IFNα2 was from Schering (USA), human IFNγ from Roussel Uclaf (Romainville, France), recombinant murine IFNα and IFNγ from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Transduction, Derivative Assay, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: IFN enhances the recruitment of UBC9 to PML NBs. A, wt MEFs and PML−/− MEFs untreated or treated with murine IFNα for 0.75 or 16 h were analyzed by confocal microscopy for UBC9 (green) and PML (red) staining. PML was stained with mouse monoclonal anti-PML antibody (clone 36.1–104, Merck millipore) that recognized murine PML. B, wt MEFs and PML−/− MEFs were untreated or treated with murine IFNα for 16 h. Cytoplasmic (C), RIPA soluble (R) and RIPA insoluble (P) fractions were analyzed by Western blotting with anti-UBC9 and anti-PML antibodies. The Coomassie brilliant blue (CBB)-stained proteins are shown. C, Expression of human PMLIII in PML−/− MEFs can recruit endogenous UBC9. Two days post-transduction with PMLIII, PML−/− MEFs were untreated or treated with murine IFNα for 16 h and analyzed by confocal microscopy for UBC9 (green) and PML (red) staining. PML was stained with mouse monoclonal anti-PML antibody (sc 966, Santa Cruz) that recognized human PML.

Article Snippet: Recombinant human IFNα2 was from Schering (USA), human IFNγ from Roussel Uclaf (Romainville, France), recombinant murine IFNα and IFNγ from R&D Systems (Minneapolis, MN).

Techniques: Confocal Microscopy, Staining, Western Blot, Expressing, Transduction