Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a-d ) Left: Super-resolution PALM intensity images of β1-WT-mEos2 (a), β1-Y795A-mEos2 (b), β3-WT-mEos2 (c), β3-Y759A-mEos2 (d) in MEFs obtained from sptPALM sequences (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labelling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labelled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). ( e ) Distributions of the diffusion coefficient D computed from the trajectories of β1-WT-mEos2 (black), β1-Y795A-mEos2 (pink), β1-Y783A-mEos2 (orange) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The grey area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. ( f ) Same as e , but with β3-WT-mEos2 (black), β3-Y759A-mEos2 (pink), β3-Y747A-mEos2 (orange). ( g ) Fraction of β1-WT and mutants undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) with or without manganese activation (Mn 2+ ). Values represent the average of the fractions obtained from different cells (error bars: SEM). ( h ) Same as g , but for β3-WT and mutants. ( i, j ) Super-resolution PALM intensity image of β1-WT (i) and β1-Y795A (j) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. (k) Kymographs generated from a sptPALM time-lapse image along the length of a FA (as shown by the dashed lines encompassing the FA in (i) and (j). ( l ) Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10-90%) of immobilization time inside FAs of β1-WT and mutants (left) and of β3-WT and mutants (right). (a-h): results for β1-WT-mEos2 without (16 cells) and with Mn 2+ (15 cells), β1-Y795A-mEos2 without (22 cells) and with Mn 2+ (22 cells), β1-Y783A-mEos2 without (16 cells) and with Mn 2+ (20 cells), β3-mEos2 without (16 cells) andx with Mn 2+ (13 cells), β3-Y759A-mEos2 without (20 cells) and with Mn 2+ (11 cells, 2 ind. exp.) and β3-Y747A-mEos2 without (16 cells) and with Mn 2+ (10 cells, 2 ind. exp.) correspond to pooled data from three independent experiments unless indicated. For (l), 6 (β1-Y795A, β1-Y783A, β3-WT), 5 (β3-Y759A, β3-Y747A) or 3 cells (β1-WT) corresponding to pooled data from three independent experiments were analysed. Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions without Mn 2+ were compared to the corresponding β-integrin-WT condition; with Mn 2+ , each given condition was compared with the value obtained without Mn 2+ . Otherwise, a black line indicates which conditions were compared. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Diffusion-based Assay, Activation Assay, Labeling, Sequencing, Generated, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a-b ) Left: Super-resolution PALM intensity images of mEos2-kindlin-2 (a) and talin-1-tdEos (b) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labelling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labelled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). ( c ) Distributions of the diffusion coefficient D computed from the trajectories of talin-1-tdEos (yellow) and mEos2-kindlin-2 (blue) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The grey area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. ( d ) Fraction of mEos2-kindlin-2 (blue) and talin-1-tdEos (yellow) undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right). Values represent the average of the fractions obtained from different cells (error bars: SEM). ( e ) Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results for mEos2-kindlin-2 (13 cells) correspond to pooled data from three independent experiments. Results for β1-WT-mEos2 (dark grey) and β3-WT-mEos2 (light grey) corresponds to data shown in and Supplementary Fig. 1. Results for talin-1-tdEos (8 cells) correspond to pooled data from two independent experiments. Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann– Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2-kindlin-2 condition. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Sequencing, Diffusion-based Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a-b ) Left: Super-resolution PALM intensity images of mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs (a) and mEos2-kindlin-2-QW614/615AA in MEFs (b) obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labelling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labelled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). ( c ) Distributions of the diffusion coefficient D computed from the trajectories of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs (black), mEos2-kindlin-2-QW614/615AA (red) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The grey area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. ( d ) Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2-kindlin-2-WT, mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs, mEos2-kindlin-2-QW614/615AA (same color-code). Values represent the average of the fractions obtained from several cells (error bars: SEM). ( e ) Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results for mEos2-kindlin-2-WT (17 cells) and mEos2-kindlin-2-QW614/615AA (33 cells) correspond to pooled data from five independent experiments. Results for mEos2-kindlin-2-WT + integrin activation by Mn 2+ (17 cells) correspond to pooled data from four independent experiments. Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2-kindlin-2-WT condition. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Sequencing, Diffusion-based Assay, Activation Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a-b ) Left: Super-resolution PALM intensity images of mEos2-kindlin-2-ΔPH (a) and mEos2-PH-domain of kindlin-2-WT (b) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labelling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labelled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). ( c ) Distributions of the diffusion coefficient D computed from the trajectories of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-ΔPH (orange) and mEos2-PH-domain of kindlin-2-WT (black) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The grey area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. ( d ) Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2-kindlin-2-WT, mEos2-kindlin-2-ΔPH and mEos2-PH-domain of kindlin-2-WT (same color-code). Values represent the average of the fractions obtained from different cells (error bars: SEM). ( e ) Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). For sptPALM data, results for mEos2-kindlin-2-WT (15 cells), mEos2-kindlin-2-ΔPH (28 cells) and mEos2-PH-domain of kindlin-2-WT (18 cells) correspond to pooled data from three independent experiments. ( f ) Fraction of proteins recruited at the membrane defined as the ratio of the membrane-level fluorescence signal (TIRF) with the total fluorescence signal of the cell (epifluorescence). For membrane fraction data, results for mEos2-kindlin-2-WT (28 cells) mEos2-kindlin-2-ΔPH (27 cells) correspond to pooled data from two independent experiments. Membrane fraction data of β3-integrin-mEos2 (17 cells), cytosolic mEos2 (13 cells), mEos2-kindlin-2-QW (15 cells), mEos2-kindlin-2-K390A (15 cells) and mEos2-kindlin-2-L357A (15 cells) correspond to one experiment. Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2-kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Sequencing, Diffusion-based Assay, Membrane, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a-e ) Top panel: Epifluorescence images of RFP-paxillin showing the localization of FAs. Bottom panel: 3D super-resolution images by DONALD displaying the axial localization of kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), paxillin ( d ) and kindlin-2-ΔPH-CAAX ( e ). For each pixel, the average axial localization of detected single molecules is color-coded. ( f ) Distributions of the single molecule axial localizations of kindlin-2-WT (blue) and paxillin (brown) inside FAs. Values represent the average of the distributions obtained from different cells. Positions of occurrence maxima (zpeak) were estimated by Gaussian fitting. ( g ) Same as f , but with kindlin-2-WT (blue), kindlin-2-QW (red), kindlin-2-ΔPH (orange) and kindlin-2-ΔPH-CAAX (green). ( h ) Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of single molecule localization inside FAs. Shown data correspond to a single experiment. kindlin-2-WT: 6 cells, kindlin-2-QW: 9 cells, kindlin-2-ΔPH: 7 cells, paxillin: 6 cells, kindlin-2-ΔPH-CAAX: 7 cells. Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2-kindlin-2-WT condition. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a-e ) Top panel: TIRF images of photoconverted mEos2 coupled to kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), kindlin-2-ΔPH-CAAX ( d ), or photoconverted mEos2 alone ( e ). Bottom panel: TIRF images of GFP-paxillin showing the localization of FAs. ( f ) Enrichment inside FAs defined as the ratio of the average fluorescence intensity inside FAs to average fluorescence intensity outside FAs. Black bars represent medians and interquartile ranges. Displayed results correspond to pooled data from two (kindlin-2-WT: 20 cells, kindlin-2-QW: 20 cells, kindlin-2-ΔPH: 20 cells, kindlin-2-ΔPH-CAAX: 20 cells) or one experiment (kindlin-2-L357A: 12 cells, kindlin-2-K390A: 12 cells, mEos2: 15 cells). Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann– Whitney rank sum test. The different conditions were compared to the mEos2-kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1101/2020.08.06.239731

Figure Lengend Snippet: ( a ) Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4h on fibronectin. Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). ( b ) Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). GFP-paxillin: 141 cells; GFP-paxillin + mEos2-kindlin-2-WT: 312 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 311 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 309 cells; GFP-paxillin + mEos2-kindlin-2-QW615/615AA: 284 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 289 cells. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). ( c ) TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of cell area ( d ) and total area of FAs ( e ) of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-ΔPH (orange) or mEos2-kindlin-2-ΔPH-CAAX (green) and 4h after seeding on fibronectin. Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 93 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 134 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 120 cells; The results correspond to pooled data from four independent experiments. Between 23 and 42 cells per experiment were used for the kindlin-2-ΔPH or kindlin-2-ΔPH-CAAX conditions (kindlin-2-WT: between 17 and 26 cells per experiment). Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01 < P < 0.05; **: 0.001 < P < 0.01; ***: 0.0001 < P < 0.001; ****: P < 0.0001.

Article Snippet: The cytosolic mEos2 construct was obtained by inserting the mEos2 sequence of the prSETa mEos2 plasmid (Addgene) in a pcDNA vector at BamHI / EcoRI endonuclease sites.

Techniques: Knock-Out, Transfection, Expressing, Negative Control, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: rsCaMPARI: an erasable marker of neuronal activity

doi: 10.1101/798298

Figure Lengend Snippet: a , rsCaMPARI expression in the Tg[ elavl3: rsCaMPARI-mRuby3] jf93 zebrafish. Scale bar is 300 μm. b , Cartoon schematic of experimental setup and image acquisition. c , Maximum intensity Z projections of the entire brain from zebrafish larvae (4 to 5 dpf) after exposure to different stimuli: no marking light, anesthetized with tricaine methanesulfonate (MS-222), freely swimming in system water, cold water (4°C), warm water (45°C), or 4-aminopyridine (4-AP). Top panels are merged reference and erased images, pseudo-colored green and magenta, respectively. Bottom panels are ΔF/F images. Scale bar is 100 μm. d , Multiple cycles of rsCaMPARI marking in the same zebrafish (5 dpf) exposed to either cold or warm water. Top panels are individual Z slices from the pallium of the same fish brain illustrating one neuron (solid white circle) that was only labeled during cold stimulus and another neuron (dashed white circle) that was variably labeled during warm stimulus. Scale bar is 10 μm. Bottom-right panel is a mean correlation matrix comparing active ensembles in the upper pallium and habenula across multiple marking cycles in the same fish.

Article Snippet: DNA constructs for pRSET_His-rsCaMPARI-mRuby3, pAAV-hsyn_NES-His-rsCaMPARI-mRuby3, pAAV-hsyn_NLS-His-rsCaMPARI-mRuby3, and pTol2-elavl3_NES-rsCaMPARI-mRuby3 are available via Addgene ( http://www.addgene.org #120804, #120805, #122092, and #122129, respectively).

Techniques: Expressing, Labeling

Journal: Journal of Immunology Research

Article Title: Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase

doi: 10.1155/2021/6629824

Figure Lengend Snippet: DNA fragment encoding Hi eno , amplification, and subcloning. (a) Agarose gel stained with ethidium bromide revealing the 1.3 kbp-sized PCR amplification products (arrow): HibATCC33930 (lane 1), NTHiBUAPPAU (lane 2), HibBUAPNAN (lane 3), amplified obtained using TOPO::HibBUAPNAN eno plasmid as a template (lane 4), NTHiBUAP96 (lane 5), and amplified obtained using TOPO::NTHiBUAP96 eno plasmid as a template (lane 6). (b) Design and construction of pRSET-A::HiENO plasmid. Lane 1: pRSET-A/XhoI digested (2.9 kbp). Lane 2: pRSET-A::HibBUAPNAN plasmid/XhoI. Lane 3: pRSET-A::HibBUAPNAN plasmid digested with XhoI and KpnI. Lane 4: pRSET-A::NTHiBUAP96 plasmid/Xho. Lane 5: pRSET-A::NTHiBUAP96 plasmid digested with XhoI and KpnI enzymes in a double restriction reaction. The product of 1.3 kbp corresponding to Hi eno gene is observed (arrow).

Article Snippet: The Hi eno DNA sequence was digested from TOPO::NTHiBUAP96 eno and TOPO:: HibBUAPNAN eno plasmids by double enzyme digestion with XhoI and KpnI and subcloned into the expression vector pRSET-A (overexpression vector, Amp r , and 6x His, Invitrogen™ by Life Technologies, Carlsbad, CA, USA), obtaining the plasmids pRSET-A::HiNTBUAP96 eno and pRSET-A::HibBUAPNAN eno ; the correct subcloning was confirmed by restriction enzyme analysis and sequencing (results not shown).

Techniques: Amplification, Subcloning, Agarose Gel Electrophoresis, Staining, Plasmid Preparation

Journal: Journal of Immunology Research

Article Title: Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase

doi: 10.1155/2021/6629824

Figure Lengend Snippet: Purification and identification of rNTHiENO. Protein was resolved by SDS-PAGE on 12% polyacrylamide gel and stained with Coomassie blue. (a) Lane M: molecular weight marker. Lane 1: E. coli Bl21 cell containing the pRSET-A::NTHiENO without IPTG induction. Lane 2: E. coli Bl21 cell containing the pRSET-A::NTHiENO with IPTG induction. Lane 3: insoluble pellet. Lane 4: washing material. Lane 5: the purified rNTHiENO. Identification of rNTHiENO by western blot using a monoclonal antibody anti-His Tag. (b) Lane M: molecular weight marker. Lane 1: pRSET-A::NTHiENO in E. coli BL21, noninduced. Lane 2: purified rNTHiENO. The band corresponding to rNTHiENO (52.0 kDa) is indicated with an arrow.

Article Snippet: The Hi eno DNA sequence was digested from TOPO::NTHiBUAP96 eno and TOPO:: HibBUAPNAN eno plasmids by double enzyme digestion with XhoI and KpnI and subcloned into the expression vector pRSET-A (overexpression vector, Amp r , and 6x His, Invitrogen™ by Life Technologies, Carlsbad, CA, USA), obtaining the plasmids pRSET-A::HiNTBUAP96 eno and pRSET-A::HibBUAPNAN eno ; the correct subcloning was confirmed by restriction enzyme analysis and sequencing (results not shown).

Techniques: Purification, SDS Page, Staining, Molecular Weight, Marker, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus *

doi: 10.1074/jbc.M115.700161

Figure Lengend Snippet: Primers used to amplify the gene encoding Cj LPMO10A The full-length protein and the truncated catalytic domain were cloned into the pRSET B expression vector using In-Fusion cloning, whereas the CBM5 and the C-terminal domain were cloned into the pNIC-CH expression vector using ligation-independent cloning. Vector overhang sequences are shown as boldface letters; the underlined sequences shows the NdeI (forward) and HindIII (reverse) restriction sites used to linearize the pRSET B vector prior to In-Fusion cloning.

Article Snippet: The amplified genes were fused into the pRSET B expression vector (linearized by the restriction endonucleases NdeI and HindIII (New England Biolabs)) using the In-Fusion HD cloning kit (Clontech).

Techniques: Clone Assay, Expressing, Plasmid Preparation, Ligation