Journal: Nature Plants

Article Title: De-repression of protein phosphatase 5 by the chaperone organizer HOP1 activates plant NLR immunity

doi: 10.1038/s41477-026-02253-4

Figure Lengend Snippet: a , LET7 PPase does not interact with TPR5G. Recombinant HIS-LET7 PPase proteins purified from E. coli were immobilized on Ni-NTA biosensor chips and incubated with soluble GST-LET7 TPR , GST-TPR5G, or GST proteins as analyte. The buffer served as a control (Ctrl). The graph shows the association (0 to 180 sec) and dissociation (180 to 360 sec) times of the interaction (left). CBB staining shows the recombinant protein expression (right). b , AlphaFold-based prediction of LET7 and its crystal structure. Structural models generated with AlphaFold 2 of full-length LET7 (left panel). The AlphaFold-predicted structure of LET7 is reminiscent of its crystal structure with the intramolecular interaction between LET7 TPR and LET7 PPase . X-ray crystal structure of LET7 showing autoinhibition of the catalytic LET7 PPase domain (green) by binding to the N-terminal TPR domain (blue) (PDB code: 7OBE) (right panel). LET7 structure figures were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 2.0, DeLano Scientific, Palo Alto, CA, 1998, https://pymol.org/2/ ). c , Potential LET7-interacting proteins identified by Y2H screens. LET7 in the pGBKT7 vector was transformed into the yeast AH109 strain. The resulting yeast transformants were then transformed with the Arabidopsis cDNA library in the pGADT7 vector, followed by screening using SD-HLT medium supplemented with 1 mM 3-AT. In-frame candidates with the number of independent colonies (count) carrying the corresponding genes are shown. d , Genotyping PCR of three hop1 mutant alleles. Genomic DNAs from WT, hop1-1 ( sail_734_f01 ), hop1-2 ( gabi_420a10 ), and hop1-3 ( salk_052232 ) were used for PCR analysis to check the annotated T-DNA insertions. The primer pair of LP and RP is used to amplify the genomic DNA fragment of HOP1 , and the primer pair of LB and RP is used to amplify the T-DNA insertions. WT seedlings were used as a negative control. e , Genotyping PCR confirms the mekk1 hop1-2 double mutant. Genomic DNAs from the F 2 generation seedlings of the heterozygous mekk1 + /- mutant crossed with hop1-2 were screened by PCR. The primer pair of LP and RP amplifies the genomic DNA fragment of HOP1 or MEKK1 , and the primer pair of LB and RP amplifies the T-DNA insertions. WT seedlings were used as a negative control. f , The rar1 mutant does not obviously suppress RNAi- MEKK1 or RNAi- BAK1/SERK4- mediated cell death. VIGS assays were performed as in Fig. using rar1-21 . Photographs were taken three weeks after inoculation. Scale bar, 1 cm. g , HOP1 TPR1 interacts with LET7 TPR in Y2H assays. Full-length or truncated versions of HOP1 and LET7 were cloned into pGBKT7 and pGADT7 vectors for Y2H assays. Experiments were performed similarly to those in Fig. . Experiments ( a , d-g ) were repeated three times with similar results.

Article Snippet: Genes cloned in recombinant protein expression vectors were induced and expressed in E. coli BL21 strain (DE3) at 16 °C using LB medium supplemented with 0.4 mM isopropyl-β- D -1-thiogalactopyranoside for 12–18 h. HIS fusion proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen), and GST fusion proteins were purified with Pierce glutathione agarose (Thermo Scientific) according to the standard protocols provided by the manufacturers.

Techniques: Recombinant, Purification, Incubation, Control, Staining, Expressing, Generated, Binding Assay, Plasmid Preparation, Transformation Assay, cDNA Library Assay, Mutagenesis, Negative Control, Clone Assay

Journal: Nature Plants

Article Title: De-repression of protein phosphatase 5 by the chaperone organizer HOP1 activates plant NLR immunity

doi: 10.1038/s41477-026-02253-4

Figure Lengend Snippet: a , Schematic diagram of mutant lines and protein motifs of HOP1. Top: T-DNA insertions in HOP1 (AT1G12270) for three mutant lines are shown with three T-DNA insertional lines. The open boxes are 5′ and 3′ UTRs, the grey boxes indicate exon protein-coding regions and the lines indicate introns. Bottom: protein motifs, including TPR1, TPR2a and TPR2b, with amino acid positions, are labelled. b – d , The hop1 mutants suppress RNAi -MEKK1 -triggered growth defects, cell death, H 2 O 2 production and PR1 gene expression. WT and hop1 plants are shown three weeks after inoculation with Agrobacterium carrying the VIGS vector targeting GFP as a control (Ctrl) or RNAi -MEKK1 ( b ). Scale bars, 1 cm. Detached leaves were stained with trypan blue for cell death or DAB for H 2 O 2 accumulation ( c ). Scale bars, 0.5 cm. The expression of PR1 was normalized to that of ACTIN2 , and the data are shown as means ± s.d. ( n = 3, biologically independent samples) ( d ). Column 1 versus Column 2, P < 0.0001, Column 1 versus Column 3, P > 0.9999, Column 3 versus Column 4, P = 0.0222. e , f , The hop1-2 mutant suppresses growth defects, cell death and the elevated expression of PR1 and PR2 in mekk1 . Three-week-old soil-grown plants are shown ( e , top) with leaves stained by trypan blue for cell death ( e , bottom). Scale bars, 1 cm. The expression of PR1 and PR2 was normalized to ACTIN2 . PR1 : Column 1 versus Column 3, P < 0.0001, Column 3 versus Column 4, P < 0.0001, Column 2 versus Column 4, P = 0.0003; PR2 : Column 1 versus Column 3, P < 0.0001, Column 3 versus Column 4, P < 0.0001, Column 2 versus Column 4, P = 0.0012. g , h , Expression of HOP1–GFP restores RNAi- MEKK1 -induced growth defects and PR1 expression in hop1-2 . HOP1 tagged with GFP under the 35S promoter ( p35S :: HOP1-GFP ) was transformed into hop1-2 . Scale bars, 1 cm ( g , top). Immunoblotting by an anti-GFP antibody shows HOP1–GFP proteins with CBB staining RBC as a loading control ( g , bottom). The expression of PR1 was normalized to ACTIN2 ( h ). Column 1 versus Column 2, P > 0.9999, Column 1 versus Column 3, P > 0.9999, Column 1 versus Column 5, P < 0.0001, Column 2 versus Column 3, P > 0.9999, Column 2 versus Column 4, P > 0.9999, Column 5 versus Column 6, P < 0.0001, Column 6 versus Column 7, P < 0.0001, Column 6 versus Column 8, P < 0.0001. i , LET7 interacts with HOP1 in Y2H assays. The experiments were performed similarly to those in Fig. . j , LET7 associates with HOP1 in BiFC assays. LET7 or HOP1 was fused with the N-terminal or C-terminal half of YFP (LET7–nYFP, LET7–cYFP, HOP1–cYFP or HOP1–nYFP) and co-expressed in N. benthamiana leaves with empty vectors carrying nYFP or cYFP (EV–nYFP or EV–cYFP) as controls. Signals were observed via confocal microscopy at 48 h post-inoculation. Scale bars, 25 μm. k , LET7 associates with HOP1 in Co-IP assays. LET7–HA was co-expressed with HOP1–GFP or GFP in protoplasts. Total proteins were immunoprecipitated with anti-HA affinity beads, followed by immunoblotting with an anti-GFP or anti-HA antibody (top two panels). Proteins before immunoprecipitation were immunoblotted and are shown as input controls (bottom two panels). l , LET7 interacts with HOP1 in pull-down assays. GST or GST–HOP1 immobilized on glutathione agarose was incubated with HIS–LET7 proteins. Top: washed beads were pelleted for immunoblotting using an anti-HIS antibody. Middle and bottom: input proteins are shown with immunoblotting before pull-down. m , LET7 interacts with HOP1 in BLI assays. Recombinant HIS–LET7 proteins were immobilized on Ni-NTA biosensor chips and incubated over a range of concentrations (1.25–10 μM) of soluble GST–HOP1 proteins as the analyte. The plot shows the association (0 to 180 s) and dissociation (180 to 360 s) times of the interaction. The equilibrium K d of the LET7 and HOP1 interaction is 2.062 ± 0.031 μM as determined by Octet BLI analysis software. The data are shown as mean ± s.d. ( n = 3 biologically independent samples in d , f and h ). Different letters indicate significant differences determined by one-way analysis of variance followed by Tukey’s test ( P < 0.05). The experiments were repeated four times in b , e , i , l and m and three times in c , d , f – h , j and k with similar results.

Article Snippet: Genes cloned in recombinant protein expression vectors were induced and expressed in E. coli BL21 strain (DE3) at 16 °C using LB medium supplemented with 0.4 mM isopropyl-β- D -1-thiogalactopyranoside for 12–18 h. HIS fusion proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen), and GST fusion proteins were purified with Pierce glutathione agarose (Thermo Scientific) according to the standard protocols provided by the manufacturers.

Techniques: Mutagenesis, Gene Expression, Plasmid Preparation, Control, Staining, Expressing, Transformation Assay, Western Blot, Confocal Microscopy, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, Recombinant, Software

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Acetylation of CDK5 at K33 causes a loss of kinase activity due to impaired ATP binding. ( a , b ) HEK293 cells were transfected with either FLAG-tagged wild type mouse CDK5 (WT), an acetyl-null mutant (K33R; KR) or a mimetic mutant (K33Q; KQ) of CDK5 in the presence of ( a ) p35-HA or ( b ) p25-HA. Lysates were immunoprecipitated (IPed) with an anti-FLAG antibody and then subjected to an in vitro phosphorylation assay using histone H1 as a substrate. The resulting phosphorylated H1 (P-H1) was visualized via immunoblot analysis (IB) with an anti-phospho-H1 antibody. Coomassie brilliant blue (CBB) staining for H1 was used as a loading control. Immunoprecipitates or whole cell lysates (WCLs) were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for WCL. ( c ) Bacterially purified, recombinant His-tagged CDK5 WT or K33-acetylated CDK5 (Ac-CDK5; Ac) was subjected to an in vitro phosphorylation assay in the presence of H1, [γ- 32 P]ATP and the indicated doses of recombinant p25. The resulting phosphorylated H1 was visualized by autoradiography. Inputs were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. ( d ) Recombinant His-CDK5 WT or His-Ac-CDK5 was incubated with or without resin conjugated to ATP. After washing, the resulting ATP-bound CDK5 was resolved by SDS-PAGE and visualized by IB with an anti-His antibody. Input signals were measured by IB with the indicated antibodies. ( e ) Recombinant His-CDK5 WT (blue-filled circles) or His-Ac-CDK5 (magenta-filled rectangles) was titrated with increasing concentration of mant-ATP. Nonlinear regression was performed to obtain a best-fit curve for a specific binding [Y = Bmax*X/(Kd + X)] and the summary of binding parameters were shown in Supplementary Table . X-axis represents the varying concentration of mant-ATP as indicated. Y-axis represents the relative fluorescence intensity of specific binding, where Bmax is maximum specific binding and Kd is equilibrium binding constant. Wilcoxon matched-pairs rank test was employed to test the binding difference between CDK5 WT and Ac-CDK5 ( ** P = 0.004; Spearman correlation coefficient, rs = 0.976; n = 3). ( f ) Lysates from HEK293 cells expressing p35-FLAG or p25-FLAG were incubated with recombinant His-CDK5 WT or His-Ac-CDK5 bound to Ni-NTA beads. Reaction mixtures were subjected to pull-down and subsequent IB with the indicated antibodies. An anti-FLAG antibody was employed to visualize the extent of CDK5-bound p35 or p25. WCLs were subjected to IB with the indicated antibodies. ( g ) Recombinant His-CDK5 WT plus increasing amounts of recombinant His-Ac-CDK5 was subjected to an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 levels were visualized by autoradiography.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Activity Assay, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, In Vitro, Phosphorylation Assay, Western Blot, Staining, SDS Page, Purification, Recombinant, Autoradiography, Incubation, Concentration Assay, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: GCN5 acetylates CDK5 at K33 in the nucleus. ( a ) Lysates obtained from HEK293 cells expressing FLAG-CDK5 plus one of the indicated KAT vectors were subjected to IP with an anti-FLAG antibody followed by IB with an anti-Ac-CDK5 antibody or anti-FLAG antibody. The intensity of the Ac-CDK5 band was measured using Image-J software and normalized to FLAG-CDK5. The fold change over the control (value = 1) is indicated at the bottom of the blot. WCLs were subjected to IB analysis with the indicated antibodies. Each KAT band is marked by the indicated letters. The asterisk indicates non-specific bands. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. *** p < 0.001; * p < 0.05; n.s, not significant. ( b ) Lysates harvested from HEK293 cells expressing FLAG-CDK5 alone or in combination with p35-MYC and/or FLAG-GCN5 were subjected to IP with an anti-FLAG antibody. The bound CDK5 was incubated in the presence of H1 and [γ- 32 P]ATP and visualized by autoradiography. The relative kinase activity of CDK5/p35 was expressed as the fold change over the control (value = 1). The bar represents the mean ± S.D from 3 independent experiments. * p < 0.05. ( c ) HEK293 cells were immunostained with an anti-Ac-CDK5 antibody. Staining specificity was confirmed by pre-incubating with the blocking peptide (EIVAL(acK)RVRLD) that was used to raise the antibody. The nuclei were counterstained with Hoechst dye. Confocal microscopy images are shown. The scale bar represents 10 μm. ( d ) HEK293 cells transfected with the indicated combinations of constructs were subjected to cellular fractionation. The resulting nuclear fractions were IPed with an anti-FLAG antibody and subsequently subjected to either IB with an anti-Ac-CDK5 or anti-FLAG antibody or an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Signals from the phosphorylated H1 were visualized by autoradiography. The fold change over the control (value = 1) is indicated. Nuclear fractions were subjected to IB with the indicated antibodies. Anti-SOD-1 and anti-lamin A/C antibodies were employed to verify the purity of the nuclear fractions.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Expressing, Software, Incubation, Autoradiography, Activity Assay, Staining, Blocking Assay, Confocal Microscopy, Transfection, Construct, Cell Fractionation, In Vitro, Phosphorylation Assay

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: SIRT1 is responsible for the deacetylation of Ac-CDK5. ( a ) HEK293 cells transiently expressing FLAG-CDK5 were treated for 24 hrs with increasing doses of nicotinamide (NA, a pan-SIRT inhibitor). Lysates were subjected to IP with an anti-FLAG antibody and probed with the indicated antibodies. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus control (value = 1) was determined. ( b ) HEK293 cells were transfected with one of the FLAG-tagged SIRTs plus FLAG-CDK5 and GCN5-HA. Lysates were subjected to IP with an anti-FLAG antibody and IB with an anti-Ac-CDK5 antibody. WCLs were subjected to IB with the indicated antibodies. Each band of SIRTs is marked by the indicated letters. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. *** p < 0.001; n.s, not significant. ( c ) FLAG-SIRT1 was expressed in HEK293 cells and purified by IP with FLAG beads. SIRT1-bound beads were incubated with recombinant His-Ac-CDK5 supplemented with β-nicotinamide adenine dinucleotide (NAD + ) to activate SIRT1. Reaction mixtures were subjected to IB with an anti-Ac-CDK5 antibody. The fold intensity of Ac-CDK5 versus the control (value = 1) was determined after normalization to the His-Ac-CDK5 inputs. The inputs were probed with the indicated antibodies. ( d , e ) HEK293 cells transfected with FLAG-CDK5 were treated with increasing doses of ( d ) EX527 (a selective SIRT1 inhibitor) or ( e ) SRT1720 (a selective SIRT1 activator) for 24 hrs. Lysates were subjected to IP with an anti-FLAG antibody followed by IB with an anti-Ac-CDK5 antibody. After normalization to FLAG-CDK5, the fold change over the control (value = 1) was determined. ( f ) Lysates were prepared from HEK293 cells transfected with FLAG-CDK5 and p35-HA and exposed to 100 μM EX527 for 24 hrs. Immunoprecipitates purified with an anti-FLAG antibody were subjected either to IB with the indicated antibodies or an in vitro phosphorylation assay in the presence of H1 and cold ATP. Phospho-H1 signals were visualized with an anti-phospho-H1 antibody. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. * p < 0.05.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Expressing, Transfection, Purification, Incubation, Recombinant, In Vitro, Phosphorylation Assay

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Pharmacological modulation of SIRT1 affects Ac-CDK5 levels and kinase activity in hippocampal neurons. ( a ) Primary cultures of hippocampal neurons were prepared from rat hippocampi isolated in gestational day 18. At the indicated days in vitro (DIV), photomicrographs were captured with an Axiovert 100. The scale bar represents 50 μm. ( b ) Cultured hippocampal neurons at DIV5 were fixed and immunostained with an anti-Ac-CDK5 antibody and an anti-NeuN antibody (a neuronal nuclear marker) followed by incubation with appropriate fluorescence-tagged secondary antibodies. Fluorescent images were obtained with an LSM700 confocal microscope. The scale bar represents 10 μm. ( c ) Hippocampal neurons at DIV3 were treated with SRT1720 or EX527 at the indicated doses for 48 hrs. Immunoprecipitates of cellular lysates purified with an anti-CDK5 antibody or IgG were subjected to IB with an anti-CDK5 antibody or an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 signals were visualized by autoradiography. After normalization to H1 or CDK5, the relative intensities of phospho-H1 and Ac-CDK5 were calculated over the controls (value = 1) and are indicated at the bottom of the blot. WCLs were subjected to IB with the indicated antibodies. ( d , e ) The relative kinase activity of CDK5 was expressed as the fold change over the control (value = 1) in the presence of ( d ) SRT1720 or ( e ) EX527. The bar represents the mean ± S.D from 4 independent experiments. *** p < 0.001. ( f ) Hippocampal neurons at DIV3 were treated with EX527 at 100 μM for 48 hrs, fixed, and then processed for immunofluorescent staining as described in ( b ). The scale bar represents 50 μm. ( g ) The fluorescence intensity of Ac-CDK5 in the nuclei of NeuN-positive neurons was measured using Image-J software. The relative fluorescence intensity was expressed as the fold change over the control (value = 1). The bars represent the mean ± S.D of 35 neurons from at least 5 randomly selected areas. *** p < 0.001.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Activity Assay, Isolation, In Vitro, Cell Culture, Marker, Incubation, Fluorescence, Microscopy, Purification, Phosphorylation Assay, Autoradiography, Staining, Software

Journal: Physiological reports

Article Title: Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

doi: 10.14814/phy2.12355

Figure Lengend Snippet: Figure 1. Characterization of IPEC-J2 cell monolayer. (A) Light microscope image of confluent cell monolayer grown on a tissue culture flask, scale bar equals 20 lm. (B) Confocal micrograph showing a top view of IPEC-J2 monolayer grown on transwell filter for 9 days, the cell borders can be distinguished by immunofluorescent staining of tight junction protein ZO-1, scale bar equals 20 lm. (C) Top view of well- differentiated IPEC-J2 cell with microvilli observed by scanning electron microscopy, figure contains one cell from cell monolayer grown on transwell filter for 9 days, scale bar equals 5 lm. (D) Progression in transepithelial electrical resistance (TEER) values of cells grown on transwell filter for 9 days. Data are given as means (SEM) of 20 separate experiments. They were assigned to experimental treatment on day 9, respectively.

Article Snippet: Images were acquired using laser scanning confocal microscopy (Nikon C-1 with Plan ApoVC 609/1.40 oil objective; Nikon EZ-C1 software; Nikon, Amsterdam, the Netherlands).

Techniques: Light Microscopy, Staining, Electron Microscopy

Journal: Physiological reports

Article Title: Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

doi: 10.14814/phy2.12355

Figure Lengend Snippet: Figure 7. Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 cells. Cell monolayers grown on transwell filters were left untreated (control) or treated with bacterial cells of ETEC (multiplicity of infection, MOI 10) alone for 3 h or pretreated with Lactobacillus spp. (MOI 100) for 6 h and then challenged by ETEC for 3 h. Monolayers stained for the tight junction protein ZO-1 (green) and nuclei stained with DAPI (blue) were detected by laser scanning confocal microscopy. The images were representatives from three separate experiments. Yellow arrows show the broken lining of ZO-1 expressions. Red circles highlight areas of cell disassociation. Scale bar equals 50 lm for all images.

Article Snippet: Images were acquired using laser scanning confocal microscopy (Nikon C-1 with Plan ApoVC 609/1.40 oil objective; Nikon EZ-C1 software; Nikon, Amsterdam, the Netherlands).

Techniques: Control, Infection, Staining, Confocal Microscopy

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: RAW macrophages transfected with mEmerald-Lifeact were fed soft deformable acrylamide- co -acrylic acid micro (DAAM)-particles (9 μm,1.4 kPa) functionalized with IgG and AF647-Cadaverineand imaged using LLSM. ( a ) Time lapse montage (min:s) of maximum intensity projections in x/y and x/z. Scale bar, 5 μm. ( b,c ) Schematic of the combined LLSM and MP-TFM experimental approach and analysis, respectively. ( d ) Front and side view of reconstructed DAAM-particle internalized in ( a ) showing target deformations and F-actin localization on particle surface. Colorscale represents the deviation of each vertex from a perfect sphere with radius equal to the median radial distance of edge coordinates to the particle centroid. Scale bar, 3 μm. Figure 1—source data 1. Numeric data for and .

Article Snippet: ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively.

Techniques: Transfection

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( a,b,c ) Violin plot shows individual phagocytic events (colored markers, n = 23), mean (black cross), and median (dashed line). ( a ) Engulfment time for individual phagocytic events. ( b ) Maximum target constriction by live-cell imaging using lattice light-sheet microscopy (LLSM). For each movie, the single time point in which target constriction was maximal was identified, and the exact constriction value was determined as described in . ( c ) Average target constriction at 50% engulfment. Average is 0.25 ± 0.04 μm (s.e.m., n = 23 cups), which is ~40% larger than observed with fixed cell data at similar stage at 0.18 ± 0.02 μm (s.e.m., n = 19) . This indicates that partial relaxation of the particle to a more spherical shape occurs during fixation, and that the fixed cell force measurements are likely a slight underestimate of the real phagocytic forces. ( d ) Volume of deformable acrylamide- co -acrylic acid-microparticles (DAAMPs) decreases with phagocytic internalization. Since hydrogel microparticles are not completely incompressible, their volume can decrease under exertion of bulk forces . Same event is shown as in . Colorscale denotes radial distance to the centroid. Time stamps are provided in min:s, and internalization is complete at the 3:58 time point. Scale bar, 3 μm. ( e ) Quantification of effective particle diameter, bulk stress, and sphericity over time for DAAMP in ( d ) Bulk compressive forces can be estimated from the previous DAAMP bulk modulus measurements (~3.8 kPa) . Gray area indicates time interval of phagocytosis. Compressive stresses arise during phagocytosis and are increased after completion of internalization. ( f ) Quantification of bulk stresses, target sphericity, and displacement of 23 live-cell phagocytic events. Compressive stresses are exerted during phagocytosis (~0.5 kPa) and increase after completion (~1.3 kPa). Particle sphericity dips during phagocytosis, but the particles return to a more spherical shape after internalization completion. Gray area indicates the duration of phagocytosis, where normalized time t = 0 indicates the start of the phagocytic event, and t = 1 internalization completion for individual events. Particle diameter and sphericity (because it could be strongly affected by imaging artifacts, see h ) were normalized to 1, using the measurements before the start of phagocytosis. ( g ) Brief F-actin accumulation (orange arrow) observed on DAAMP phagosome following internalization. Time lapse montage (min:s) of RAW macrophage transfected with mEmerald-Lifeact internalizing a DAAMP (9 μm, 6.5 kPa) functionalized with IgG and AF647-Cadaverine and imaged using LLSM. Scale bar, 5 μm. (h) Typical LLSM images showing artifacts that hinder particle reconstruction and force analysis. Artifacts are not obvious in the xy-plane, but a strong ‘striping’ artifact, with strong fluctuations in fluorescent intensity (yellow arrows) are visible along the optical axis. Shape reconstruction in microparticle traction force microscopy (MP-TFM) is critically dependent on particle edge detection, which shows clear irregularities (yellow arrows) because of the striping artifact. Such artifacts result in large apparent particle deformations inside and outside the cell-target contact area, and can make it impossible to converge on a solution when solving the elasticity theory problem to infer tractions from target deformations. Because of this, we only used the confocal image data for force analysis. Scale bar, 5 μm.

Article Snippet: ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively.

Techniques: Live Cell Imaging, Microscopy, Imaging, Transfection

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( a ) Time lapse montage (min:s) of RAW macrophage transfected with mEmerald-Lifeact internalizing a deformable acrylamide- co -acrylic acid-microparticle (DAAMP) (9 μm, Young’s modulus 6.5 kPa) functionalized with IgG and AF647-Cadaverine and imaged using lattice light-sheet microscopy (LLSM). Maximum intensity projections in x/y are shown. Scale bar, 5 μm. ( b ) Side view of reconstructed DAAMP internalized in ( a ) showing target deformations and F-actin localization on particle surface. Arrows point at loci of F-actin accumulation and protrusion into the target, similar to those observed for 1.4 kPa particles . Scale bar, 3 μm. ( c ) Average deformation and F-actin intensity profiles along the phagocytic axis to the cup rim for fixed RAW cells imaged using confocal microscopy. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets (in 30 bins). Only targets beyond 40% engulfment were included (n = 28, 17 for 1.4 and 6.5 kPa particles, respectively). ( d ) Target sphericity. Violin plots show individual phagocytic events (markers) (n = 33, 19 for 1.4 and 6.5 kPa particles, respectively), mean (black cross) and median (dashed line). ***Two-sided Wilcoxon rank sum test: p = 5.9 × 10 –6 . ( e ) Analysis of constriction magnitude for all phagocytic events (n = 33, 19 for 1.4 and 6.5 kPa particles, respectively). Individual markers indicate individual measurements, lines indicate averages within the five bins. **Two-sided Wilcoxon rank sum test: p < 0.01. Unsurprisingly, the average target constriction is higher for 1.4 kPa targets (170 ± 25 nm, s.e.m.) than for 6.5 kPa targets (70 ± 15 nm, s.e.m.). Modeling this constriction as a simple cylindrical indentation into a flat surface ( F ≈ 4 π E d 9 ), where F is the force, E is the target Young’s modulus, and d is the target Young’s modulus, rough force estimates can be obtained. With ~7 and ~13 nN for the 1.4 and 6.5 kPa targets, respectively, moderately higher total contractile forces are observed on the stiffer targets. All error bars indicate s.e.m. ( f ) Phagocytic efficiency upon drug treatment follows the same trends as for softer particles (see and ). Efficiency was evaluated as the number of internalized particles divided by the total number of cell-associated particles. Uptake was evaluated 15 min after addition of particles and normalized to internalization by DMSO-treated cells. Two independent experiments were performed where 40–200 particles were measured per condition for each experiment. *p = 0.04 (t-test result for hypothesis, mean = 1).

Article Snippet: ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively.

Techniques: Transfection, Microscopy, Confocal Microscopy

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( a ) Time lapse montage (min:s) of a BMDM transfected with mEmerald-Lifeact internalizing a soft deformable acrylamide- co -acrylic acid-microparticle (DAAMP) (9 μm, Young’s modulus 0.3 kPa) functionalized with IgG and AF647-Cadaverine and imaged using lattice light-sheet microscopy (LLSM). Maximum intensity projections in x/z are shown. Softer targets were chosen than those for RAW cells, because BMDMs induced less strong target deformations. Scale bar, 5 μm. ( b ) Side view of reconstructed DAAMP internalized in ( a ) showing target deformations and F-actin localization on particle surface. Similar spots of F-actin accumulation and protrusion into the target can be observed as for RAW cells engulfing 1.4 or 6.5 kPa particles ( and , ). Scale bar, 3 μm.

Article Snippet: ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively.

Techniques: Transfection, Microscopy

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet:

Article Snippet: ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively.

Techniques: Recombinant, Plasmid Preparation, Construct, Concentration Assay, Transfection, Software

Journal: Nutrients

Article Title: Cell Membrane Fatty Acids and PIPs Modulate the Etiology of Pancreatic Cancer by Regulating AKT

doi: 10.3390/nu17010150

Figure Lengend Snippet: Dietary PUFAs modify PIP3 localization in the membrane, affecting AKT signaling, and exogenous PUFA administration by lipid nanoparticles (LNPs) improves PUFA delivery to the membranes. ( A ) Representative images of Panc-1 cells expressing the PH-BtK-EGF fusion protein (PIP3 biosensor) and incubated with 40 µM of DHA and LA. GFP (PIP3) expression was assessed with a confocal microscope. White arrows point to GFP-enriched spots at the plasma membrane. ( B ) Percentage of cells with membrane-positive staining relative to the total number of green cells (4 different fields of view, 20×). ( C ) Panc-1 cells transfected with GFP-C1-PLCdelta-PH were incubated with DHA and LA 40 µM for 48 h and subjected to immunoprecipitation. Western blot images of PI3k-alpha pulled down with GFP antibody to assess the binding of PI3K to PIP3. ( D ) Quantification of the replicates performed by immunoblotting. Quantification was done using ImageJ (NIH) software. All the images are representative of the averaged results of the scoring (n = 2). ( E ) The Panc-1 cell line was incubated with a lipid nanoparticle formulation (LNP) consisting of 90% GMO and 10% cholesterol with 40 µM DHA. The BSA group represents the control group treated with DHA bound to BSA (BSA.DHA) as a carrier. The LNP group represents the group treated with DHA encapsulated in LNP (LNP-DHA). Western blot images probing for total and phosphorylated AKT proteins. GAPDH was used as a loading control. ( F ) Quantification of the replicates performed by immunoblotting relative to the GAPDH expression level. ( G ) Representative images of confocal microscopy of Panc-1 cells treated with the complex BSA-DHA and with the complex LNP-DHA. Fatty acids are shown in red, LNP are shown in green, and the nucleus in blue. Asterisks in the graphs above a group define significance against the normal diet control: * p < 0.05; *** p < 0.0005. Bars indicate significant differences between two groups. Results are expressed as the mean ± standard deviation (SD).

Article Snippet: The GFP-C1-PLCdelta-PH vector (encodes a PIP2 lipid-selective PH domain that can be used as a fluorescent translocation biosensor to monitor changes or local differences in the concentration of plasma membrane PIP2 lipids) was a gift from Tobias Meyer (Addgene plasmid #21179).

Techniques: Membrane, Expressing, Incubation, Microscopy, Staining, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Software, Formulation, Control, Confocal Microscopy, Standard Deviation

Journal: eLife

Article Title: RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells

doi: 10.7554/eLife.63452

Figure Lengend Snippet:

Article Snippet: GST-fusion RNF41 recombinant proteins were expressed in bacterial BL21 DE3 E. coli (Promega) and purified using Glutathione-Sepharose resin (GE Healthcare) and SEC.

Techniques: Expressing, Transgenic Assay, Transfection, Construct, Recombinant, Synthesized, Confocal Microscopy, Plasmid Preparation, shRNA, Sequencing, Positive Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Bicinchoninic Acid Protein Assay, Microarray, Labeling, In Situ, Software

Journal: Biomacromolecules

Article Title: Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs

doi: 10.1021/acs.biomac.7b01196

Figure Lengend Snippet: Structure of PhMV and the strategies used for the functionalization of PhMV-derived VLPs. (A) Ribbon diagram of the PhMV VLP icosahedral asymmetric unit consisting of A, B, and C subunits. Five A subunits make up pentameric capsomeres at the icosahedral five-fold axes, whereas B and C subunits form hexamers at the icosahedral three-fold axes. The A subunit is shown in black, the B subunit is shown in red, and the C subunit is shown in pink. Representation of internal and external surfaces (with 180° rotation) of the PhMV asymmetric unit, highlighting surface-exposed (K62, K143, K153, and K166) and buried (K8, K10, K76, K182, and K127) lysine residues (blue), and the single cysteine residue (C75, green). PhMV forms from 180 identical coat protein subunits arranged in a T = 3 icosahedral structure. Images created using UCSF Chimera (PDB: 1E57). The capsid is characterized by prominent protrusions of pentamers and hexamers. (B) Schematic of PhMV labeling with sulfo-Cy5 NHS ester (blue) using lysine-NHS ester chemistry. (C) Conjugation of Cy5.5-maleimide (pink) to internal cysteine residues using maleimide–thiol chemistry. (D) DOX (red) infusion into PhMV, leading to cargo-loaded particles. Washing and ultracentrifugation are used to remove excess DOX, yielding intact PhMV with infused DOX (DOX-PhMV). (E) Schematic of PS (blue) loading into PhMV via infusion, yielding PS-PhMV particles.

Article Snippet: Avidin Agarose Affinity Binding Assay Biotinylated VLPs and controls were tested for their ability to bind avidin agarose resin (Pierce).

Techniques: Derivative Assay, Residue, Labeling, Conjugation Assay

Journal: Biomacromolecules

Article Title: Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs

doi: 10.1021/acs.biomac.7b01196

Figure Lengend Snippet: Characterization of fluorophore-labeled and drug-loaded VLPs. (A) SDS-PAGE analysis of PhMV-KE-Cy5, PhMV-CI-Cy5.5, PS-PhMV, and DOX-PhMV visualized under UV light (UV), white light (WL) before staining, and under white light after Coomassie blue staining (CS). M = SeeBlue Plus2 molecular weight (kDa) standard; 1 = Native PhMV; 2 = PhMV-KE-Cy5; 3 = PhMV-CI-Cy5.5; 4 = PS-PhMV; 5 = PS; 6 = DOX-PhMV; 7 = DOX. (B) Agarose gel electrophoresis of PhMV-KE-Cy5, PhMV-CI-Cy5.5, PS-PhMV, and DOX-PhMV visualized under UV light and white light before, and under white light after Coomassie blue staining (CS). Functionalized particles loaded in each lane was as described above for SDS-PAGE analysis (note: the white marks in the center of the gel are the pockets into which the samples were loaded prior to electrophoretic separation). (C) Size exclusion chromatograms of PhMV-KE-Cy5 [monitored at 260 nm (blue), 280 nm (red), and 646 nm (green), sulfo-Cy5 NHS ester absorbance], PhMV-CI-Cy5.5 [monitored at 260 nm (blue), 280 nm (red), and 673 nm (green), Cy5.5-maleimide absorbance], PS-PhMV [monitored at 260 nm (blue), 280 nm (red), and 450 nm (green), PS absorbance] and DOX-PhMV [monitored at 260 nm (blue), 280 nm (red), and 496 nm (green), DOX absorbance]. (D) Transmission electron micrographs of negatively stained (UAc) PhMV-KE-Cy5, PhMV-CI-Cy5.5, PS-PhMV, and DOX-PhMV.

Article Snippet: Avidin Agarose Affinity Binding Assay Biotinylated VLPs and controls were tested for their ability to bind avidin agarose resin (Pierce).

Techniques: Labeling, SDS Page, Staining, Molecular Weight, Agarose Gel Electrophoresis, Transmission Assay

Journal: Biomacromolecules

Article Title: Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs

doi: 10.1021/acs.biomac.7b01196

Figure Lengend Snippet: Characterization of PhMV-biotin conjugates. (A) Biotinylated PhMV particles separated by denaturing SDS-PAGE visualized after staining with Coomassie. M = SeeBlue Plus2 molecular weight marker. (1) Native PhMV; (2) PhMV-CI-bio; (3) PhMV-KE-bio. (B) Biotinylated PhMV particles separated by agarose gel electrophoresis visualized after Coomassie staining. (C) Flow through and eluted biotinylated particles from avidin bead binding assay separated by SDS-PAGE and stained with Coomassie. (4) Native PhMV flow through; (5) PhMV-KE-bio flow through; (6) PhMV-CI-bio flow through; (7) bound native PhMV; (8) bound PhMV-KE-bio; (9) bound PhMV-CI-bio. (D) Avidin bead assay: PhMV samples are exposed to avidin-coated beads; only particles with biotin on the external surface bind to the beads.

Article Snippet: Avidin Agarose Affinity Binding Assay Biotinylated VLPs and controls were tested for their ability to bind avidin agarose resin (Pierce).

Techniques: SDS Page, Staining, Molecular Weight, Marker, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Binding Assay

Journal: Biomacromolecules

Article Title: Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs

doi: 10.1021/acs.biomac.7b01196

Figure Lengend Snippet: Cell uptake studies with fluorescence-labeled PhMV analyzed by confocal microscopy and FACS. (A) Confocal images representing the internalization of PhMV-KE-Cy5 by A2780, MDA-MB-231, and PC-3 cells. PhMV was tagged with sulfo-Cy5 NHS ester (pseudo green), the cell membrane was stained with wheat germ agglutinin (WGA)-Alexa Fluor 555 (pseudo pink), and the nucleus was stained with DAPI (blue). Scale bars = 25 μm. (B) Flow cytometry of A2780, MDA-MB-231, and PC-3 cells following 6 h incubation with PhMV-KE-Cy5 particles. Inset provides mean fluorescence intensities (MFIs) of cells for each sample quantified from three replicates with standard deviations (±) in the corresponding cell panels; blue = cells only, red = after incubation with VLPs. (C) Confocal imaging of A2780 and MDA-MB-231 cells showing colocalization of PhMV-CI-Cy5.5 particles with the endolysosomal marker LAMP-1 after 6 h. Nuclei are shown in blue, endolysosomes are stained with mouse antihuman LAMP-1 antibody (red) and PhMV-CI-Cy5.5 (pseudo green). Colocalization signals are shown in white (overlay, bottom panel). Scale bars = 25 μm. (D) FACS quantification of PhMV-CI-Cy5.5 uptake using A2780, MDA-MB-231, PC-3, HeLa, U87, HT1080, RAW 264.7, and NIH/3T3 cells. Inset provides mean fluorescence intensities (MFIs) of cells for each sample quantified from three replicates with standard deviations (±) in the corresponding cell panels; blue = cells only, red = after incubation with VLPs. All samples were measured in triplicates and analyzed using FlowJo software.

Article Snippet: Avidin Agarose Affinity Binding Assay Biotinylated VLPs and controls were tested for their ability to bind avidin agarose resin (Pierce).

Techniques: Fluorescence, Labeling, Confocal Microscopy, Membrane, Staining, Flow Cytometry, Incubation, Imaging, Marker, Software

Journal: Frontiers in immunology

Article Title: A non-invasive nanobody probe for high precision mapping of Lck spatial distribution.

doi: 10.3389/fimmu.2024.1440499

Figure Lengend Snippet: FIGURE 1 Development of Lck binding Nbs. (A) In-cell screening assay for the identification of the most potent binder. Constitutively expressed Lck and individual Nbs inducibly expressed under the control of Dox, were transiently cotransfected in HEK293 cells. Postnuclear lysates were subjected to immunoprecipitation using anti-HA beads and Nb-bound Lck was revealed by western blotting, as indicated. Left panel. Representative experiment screening the Lck-binding capacity of four different Nbs, as indicated. Cells cultured in the absence of Dox (-Dox sample) were used as a control for the anti-HA IPs. Corresponding blots of total cell lysates reveal expression levels of Nb and Lck constructs in the samples. The bar graph presents collective data from the screening of all 30 different anti-Lck Nbs as indicated. Immunoprecipitation potency was quantitated by densitometric analysis of the Lck bands in the immunoprecipitation blots, normalized for individual Nb expression levels as revealed by anti-HA blots of the same membranes. (B) Selectivity assessment of NbGT for Lck. Inducibly expressed LckWT and the indicated mutant or chimeric forms, together with NbGT were transiently cotransfected in HEK293 cells. Postnuclear lysates were subjected to immunoprecipitation as in (A) Corresponding blots of total cell lysates ensure equal expression of NbGT and Lck constructs in all samples.

Article Snippet: HA-Tag (C29F4) Rabbit mAb (Sepharose® Bead Conjugate) (Cat: 3956) used for Immunoprecipitation, Mouse anti-HA (Cat: 2367) used for western blotting, Rabbit anti-pSrc (Y416), detecting Lck pY394 (Cat: 2101), Rabbit anti-phospho-Lck (Y505) (Cat: 2751) antibodies were purchased from Cell Signaling Technology (CST).

Techniques: Binding Assay, Screening Assay, Control, Immunoprecipitation, Western Blot, Cell Culture, Expressing, Construct, Mutagenesis

Journal: Frontiers in immunology

Article Title: A non-invasive nanobody probe for high precision mapping of Lck spatial distribution.

doi: 10.3389/fimmu.2024.1440499

Figure Lengend Snippet: FIGURE 2 NbGT is a is a highly precise probe for Lck in intact cells. (A) Schematic representation of N-terminally modified NbGT constructs used in this study. Myristoylated (Glycine, G) and Palmitoylated (Cysteine, C) residues are in bold and underlined. (B, C) N-terminal modifications do not compromise NbGT binding to Lck. (B) Constitutively expressed Lck and the indicated N-terminally modified NbGT constructs were transiently cotransfected in HEK293 cells. Postnuclear lysates were subjected to immunoprecipitation as in Figure 1A. Western blots of total cell lysates ensure equal expression of NbGT and Lck constructs in all samples. (C) Representative confocal microscopy imaging of HEK293 cells co-transfected with Lck (red) and CD4- NbGT (green) as indicated. Lck staining of Jurkat cells right-hand side panel serves as a means of comparison of the subcellular distribution between endogenous (Jurkat) and exogenously overexpressed proteins (HEK 293). Scale bar, 10µm. (D) Expression of N-terminally modified NbGT constructs in Jurkat cells does not affect Lck regulation. Stable Jurkat cell lines expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were stained with antibodies against phosphorylated forms of the activating (Y394) and inhibitory (Y505) tyrosines and analyzed by FACS. MFI values for each antibody staining were determined by analysis with the Flowjo software. Graphs show collective data from three independent experiments (Unpaired Student t test; mean +/- standard deviation [SD]; ns: not significant). (E) Expression of N-terminally modified NbGT constructs in Jurkat cells does not compromise the physiological function of Lck. Stable Jurkat cell lines expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were either left untreated or were stimulated with anti-CD3ϵ for 2min at 37°C. Samples were stained with an antibody recognizing the phosphorylated form of the TCR z chain ITAM tyrosine 142 (pY142) and analysed by FACS. The Graph depicts frequency of cells positive for Y142 phosphorylation from three independent experiments (Unpaired Student t test; mean +/- SD; ns: not significant). (F) N-terminally modified NbGT constructs colocalize with endogenous Lck. Jurkat cells expressing the indicated N-terminally modified NbGT forms were immobilized on poly-D-lysine coated microscope slides, stained for Lck (red) and HA (green), and visualized by confocal microscopy. Colocalization of Lck and NbGT was quantified by the coloc2 pre-installed plugin of Image J Collective colocalization analysis results from two independent experiments are displayed on the adjacent graph. Number of cells for each sample ≥30. Scale bar, 5µm (Unpaired Student t test; mean +/- SD; ns, not significant, ****P < 0.0001). (G) Gradation of Lck binding amongst the different N-terminally modified NbGT constructs. Post nuclear lysates of Jurkat cells expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were subjected to immunoprecipitation using anti-HA beads and Nb-bound Lck was revealed by western blotting as in Figure 1A. Western blots of total cell lysates ensure equal expression of Lck and NbGT constructs in all samples. Immunoprecipitation potency was quantitated by densitometric analysis of the immunoprecipitated Lck bands, normalized for individual Nb expression levels as revealed by a-HA blots. Collective data from 4 independent experiments is depicted in the adjacent bar graph. (Unpaired Student t test; mean +/- SD; **P < 0.01, ****P < 0.0001).

Article Snippet: HA-Tag (C29F4) Rabbit mAb (Sepharose® Bead Conjugate) (Cat: 3956) used for Immunoprecipitation, Mouse anti-HA (Cat: 2367) used for western blotting, Rabbit anti-pSrc (Y416), detecting Lck pY394 (Cat: 2101), Rabbit anti-phospho-Lck (Y505) (Cat: 2751) antibodies were purchased from Cell Signaling Technology (CST).

Techniques: Construct, Binding Assay, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Imaging, Transfection, Staining, Comparison, Software, Standard Deviation, Phospho-proteomics, Microscopy

Journal: The Journal of Cell Biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: APC-m4 alters actin dynamics at FAs. U2OS cells were depleted of endogenous APC and rescued with refractory APC constructs (APC-WT or APC-m4) along with plasmids expressing GFP-actin and mCherry-zyxin. (A) FRAP analysis, in which ROI were selected where GFP-actin and mCherry-zyxin signals overlap (see orange box in cartoon). ROIs were then bleached and monitored for GFP-actin fluorescence recovery. Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three independent experiments ( n = 30 ROIs from n = 15 cells per condition). (B) FRAP experiments as in A except that ROIs were selected along stress fibers at a distance (>5 µm) from FAs (see orange box in cartoon). Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three independent experiments ( n = 30 ROIs from n = 15 cells per condition). (C) Average time to 50% maximal recovery for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (D) Average immobile fraction (does not recover in observation window) for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: *, P < 0.05. (E) Average time to 50% maximal recovery for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (F) Average immobile fraction for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant.

Article Snippet: For immunoprecipitations ( and Fig. S5 E), cells were transfected as described above with plasmids expressing full-length APC (WT or m4), GFP-LC3 (11546; Addgene), GFP-LAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Construct, Expressing, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: APC-m4 decreases the levels and/or densities of key molecular components at FAs. All data are from MDA-MB-231 cells expressing APC constructs (APC-WT or APC-m4), using fixed or live cell imaging as indicated. (A) Representative immunostaining of endogenous active phospho-Src detected by confocal imaging. Scale bar, 40 µm. Green boxed regions correspond to zoom panels (right; scale bar, 10 µm), which highlight the localization of phospho-Src at the cell periphery. (B) Representative immunostaining of endogenous active phospho-paxillin detected by confocal imaging. Scale bar, 25 µm. Green boxed regions correspond to zoom panels (right; scale bar, 5 µm), which highlight the localization of phospho-paxillin at the cell periphery. (C) Representative immunostaining of endogenous active phospho-FAK tyrosine kinase detected by confocal imaging. Scale bar, 25 µm. Green boxed regions correspond to zoom panels (right; scale bar, 5 µm), which highlight the localization of phospho-FAK at the cell periphery. (D) Densities of endogenous phospho-Src, phospho-paxillin, and phospho-FAK determined from cell images as in A–C. Data averaged from three independent experiments. n ≥ 56 cells for phospho-Src, n = 35 cells for phospho-paxillin, and n ≥ 103 cells for phospho-FAK per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (E) Representative SIM images of cells immunostained for phospho-Src (green) and paxillin (pink). Scale bar, 5 µm. White boxed regions correspond to zoom panels (right; scale bar, 2 µm), highlighting the localization of phospho-Src and paxillin at FAs. (F) Representative SIM images of cells immunostained for phospho-paxillin (green) and paxillin (pink). Scale bar, 5 µm. White boxed regions correspond to zoom panels (right; scale bar, 2 µm), highlighting the localization of phospho-paxillin and paxillin at FAs. (G) Representative SIM images of cells immunostained for phospho-FAK (green) and paxillin (pink). Scale bar, 5 µm. White boxed regions correspond to zoom panels (right; scale bar, 2 µm), highlighting the localization of phospho-paxillin and paxillin at FAs. (H) Density of phospho-Src, phospho-paxillin, and phospho-FAK staining at individual FAs from cell images as in E–G. Data averaged from two independent experiments. n = 50 FAs total from 15 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001; **, P < 0.01. (I) Densities of signals at FAs for different components: GFP-paxillin and mCherry-zyxin densities were measured from cell images captured by TIRF microscopy; vinculin densities were measured from immunofluorescence images captured by confocal microscopy. Data averaged from three independent experiments. n = 34–159 FAs from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test : ****, P < 0.0001; ***, P < 0.001.

Article Snippet: For immunoprecipitations ( and Fig. S5 E), cells were transfected as described above with plasmids expressing full-length APC (WT or m4), GFP-LC3 (11546; Addgene), GFP-LAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Expressing, Construct, Live Cell Imaging, Immunostaining, Imaging, MANN-WHITNEY, Two Tailed Test, Staining, Microscopy, Immunofluorescence, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: APC-m4 alters autophagosome dynamics at FAs. All data are from live cell TIRF imaging of migrating MDA-MB-231 cells expressing APC constructs (APC-WT or APC-m4), along with markers for autophagosomes (GFP-LC3) and FAs (mCherry-zyxin). For all panels, data are averaged from at least three experiments. (A) Representative time-lapse imaging showing autophagosomes (GFP-LC3, cyan) and FAs (mCherry-zyxin, pink). Time = 0 corresponds to maximum mCherry-zyxin fluorescence intensity (peak FA growth). Scale bar, 3 µm. (B) Percentage of mature FAs targeted by autophagosomes, analyzed from experiments as in A. n > 800 FAs per condition from n ≥ 20 cells per condition. Error bars, SEM. Statistical significance calculated by one-way ANOVA Dunn’s multiple comparisons test: n.s., not significant. (C) Histograms showing distributions of mature FAs targeted by autophagosomes in the 30-min observation window, from experiments as in A. n = 40 FAs from n > 5 cells per condition. (D) Scatter plot showing dwell times of autophagosomes at FAs, analyzed from experiments as in A. n ≥ 42 autophagosomes per condition from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (E) Scatter plot showing time after first appearance of an autophagosome at the FA to complete FA disassembly, analyzed from experiments as in A. n = 31 FAs (APC-WT) or n = 50 FAs (APC-m4) from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (F) Overlaid histograms showing time elapsed from peak FA maturity (time = 0) to arrival of autophagosome during the 40-min observation window. Negative numbers correspond to rare events in which autophagosomes arrive before FA peak maturation. Data are analyzed from experiments as in A. n = 20 FAs from n > 5 cells per condition. (G) Percentage of mature FAs targeted by GFP-NBR1 receptor, analyzed from live imaging experiments as in A, except using cells expressing mCherry-zyxin and GFP-NBR1. n = 100 FAs (from 15 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (H) Scatter plot showing dwell times of GFP-NBR1 interactions with FAs, analyzed from live imaging experiments as in G. n = 40 GFP-NBR1 visits to FAs (from n = 10 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (I) Coimmunoprecipitation of endogenous NBR1 with GFP-LC3, pulled down using GFP–Trap-A agarose beads. Cells transfected with empty vector (expressing GFP alone instead of GFP-LC3) serve as a negative control.

Article Snippet: For immunoprecipitations ( and Fig. S5 E), cells were transfected as described above with plasmids expressing full-length APC (WT or m4), GFP-LC3 (11546; Addgene), GFP-LAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Imaging, Expressing, Construct, Fluorescence, MANN-WHITNEY, Two Tailed Test, Transfection, Plasmid Preparation, Negative Control

Journal: The Journal of Cell Biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: Key resources

Article Snippet: For immunoprecipitations ( and Fig. S5 E), cells were transfected as described above with plasmids expressing full-length APC (WT or m4), GFP-LC3 (11546; Addgene), GFP-LAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Virus, Recombinant, Sequencing, Plasmid Preparation, Software, Fluorescence, Imaging