Structured Review

Bio-Rad imagelab software
Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. <t>ImageLab</t> software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P
Imagelab Software, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/imagelab software/product/Bio-Rad
Average 99 stars, based on 40 article reviews
Price from $9.99 to $1999.99
imagelab software - by Bioz Stars, 2022-09
99/100 stars

Images

1) Product Images from "Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro"

Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6646

Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P
Figure Legend Snippet: Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P

Techniques Used: Western Blot, Expressing, Transfection, Software

2) Product Images from "Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance"

Article Title: Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkaa974

p53's N-terminus as binding partner for POLι. K562 cells were transfected with expression plasmids for p53(WT), p53(22Q/23S) or empty vector (ctrl). Immunoprecipitations were performed 48 h after transfection. Pull-downs engaged polyclonal rabbit POLι-antibody (Bethyl, A301–304A) or control rabbit IgG, subsequent immunoblotting relied on anti-POLι, anti-p53 (mix of mAbs Pab421 and DO1) and light chain-specific peroxidase-coupled secondary antibody. The left panel shows a representative Western Blot. The right panel shows the quantification of three immunoprecipitations. Quantification of p53 was carried out with ImageLab software and corrected for values of the pull-down protein POLι, whereby the mean value for p53(WT) samples was set to 1. Mean ±SD. Note, that the values of the ctrl were subtracted from the values of p53(WT) and p53(22Q/23S). Due to the small number of values (n = 3) no statistical significant differences were achieved when comparing p53(WT) to p53(22Q/23S) or ctrl. IP = Immunoprecipitation of POLι.
Figure Legend Snippet: p53's N-terminus as binding partner for POLι. K562 cells were transfected with expression plasmids for p53(WT), p53(22Q/23S) or empty vector (ctrl). Immunoprecipitations were performed 48 h after transfection. Pull-downs engaged polyclonal rabbit POLι-antibody (Bethyl, A301–304A) or control rabbit IgG, subsequent immunoblotting relied on anti-POLι, anti-p53 (mix of mAbs Pab421 and DO1) and light chain-specific peroxidase-coupled secondary antibody. The left panel shows a representative Western Blot. The right panel shows the quantification of three immunoprecipitations. Quantification of p53 was carried out with ImageLab software and corrected for values of the pull-down protein POLι, whereby the mean value for p53(WT) samples was set to 1. Mean ±SD. Note, that the values of the ctrl were subtracted from the values of p53(WT) and p53(22Q/23S). Due to the small number of values (n = 3) no statistical significant differences were achieved when comparing p53(WT) to p53(22Q/23S) or ctrl. IP = Immunoprecipitation of POLι.

Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Software, Immunoprecipitation

3) Product Images from "Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food"

Article Title: Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food

Journal: Food Chemistry: Molecular Sciences

doi: 10.1016/j.fochms.2022.100126

Effect of leaves EtAc and ButOH extracts on LDLR and PCSK9 expression in Huh7 cell line. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p
Figure Legend Snippet: Effect of leaves EtAc and ButOH extracts on LDLR and PCSK9 expression in Huh7 cell line. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p

Techniques Used: Expressing, Western Blot, Software, Quantitative RT-PCR

Effect of fruit extract on LDLR and PCSK9 expression in Huh7 cells. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C) and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p
Figure Legend Snippet: Effect of fruit extract on LDLR and PCSK9 expression in Huh7 cells. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C) and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p

Techniques Used: Expressing, Western Blot, Software, Quantitative RT-PCR

4) Product Images from "Architecture of the chikungunya virus replication organelle"

Article Title: Architecture of the chikungunya virus replication organelle

Journal: bioRxiv

doi: 10.1101/2022.04.05.487153

Representative gels related to Fig. 3 . (A-C). Copelletation of nsP1 with multilamellar vesicles (MLVs) with varying percentages of the anionic phospholipids POPG (A), PI(4)P (B), and PI(4,5)P 2 (C) in a background of POPC and 20% cholesterol. The supernatant and pellet were run on 10% SDS-PAGE. After destaining the Coomassie stained gel, image was acquired with a Chemidoc Imaging System (Bio-Rad) and the relative intensity of bands were quantified using ImageLab software (Bio-Rad) and plotted as shown in Figure 3 (A-C) .
Figure Legend Snippet: Representative gels related to Fig. 3 . (A-C). Copelletation of nsP1 with multilamellar vesicles (MLVs) with varying percentages of the anionic phospholipids POPG (A), PI(4)P (B), and PI(4,5)P 2 (C) in a background of POPC and 20% cholesterol. The supernatant and pellet were run on 10% SDS-PAGE. After destaining the Coomassie stained gel, image was acquired with a Chemidoc Imaging System (Bio-Rad) and the relative intensity of bands were quantified using ImageLab software (Bio-Rad) and plotted as shown in Figure 3 (A-C) .

Techniques Used: SDS Page, Staining, Imaging, Software

5) Product Images from "PCSK9 Induces Rat Smooth Muscle Cell Proliferation and Counteracts the Pleiotropic Effects of Simvastatin"

Article Title: PCSK9 Induces Rat Smooth Muscle Cell Proliferation and Counteracts the Pleiotropic Effects of Simvastatin

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22084114

Effect of PCSK9 overexpression on Ldlr in SMCs. SMC puro and SMC PCSK9 were seeded in DMEM/10% FCS (3 × 10 5 cells/well in a 6-well tray). The day after, the medium was changed with DMEM containing either a high (10%) or low (0.4%) concentration of FCS and, after an additional 72 h, the total cell lysates were prepared. ( A ) PCSK9 and Ldlr protein expression were evaluated by Western blot analysis. α-tubulin was used as the loading control. Panel A shows representative images of three independent experiments. ( B ) Densitometric readings for the Ldlr were evaluated using ImageLab software. Ldlr, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SMC PCSK9 , smooth muscle cells overexpressing PCSK9. Differences between conditions were assessed by Student’s t -test and one-way ANOVA (when necessary). * p
Figure Legend Snippet: Effect of PCSK9 overexpression on Ldlr in SMCs. SMC puro and SMC PCSK9 were seeded in DMEM/10% FCS (3 × 10 5 cells/well in a 6-well tray). The day after, the medium was changed with DMEM containing either a high (10%) or low (0.4%) concentration of FCS and, after an additional 72 h, the total cell lysates were prepared. ( A ) PCSK9 and Ldlr protein expression were evaluated by Western blot analysis. α-tubulin was used as the loading control. Panel A shows representative images of three independent experiments. ( B ) Densitometric readings for the Ldlr were evaluated using ImageLab software. Ldlr, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SMC PCSK9 , smooth muscle cells overexpressing PCSK9. Differences between conditions were assessed by Student’s t -test and one-way ANOVA (when necessary). * p

Techniques Used: Over Expression, Concentration Assay, Expressing, Western Blot, Software

6) Product Images from "Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication"

Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

Journal: Heliyon

doi: 10.1016/j.heliyon.2018.e00525

A-E . The relative intensities of protein bands (mean ± SE) are shown in bar graphs for TLR4, connexin 43 (Cx 43), Na + /K + ATPase, GLT-1, GLAST-1. Intensity were quantified using the ImageLab software in the linear exposure range and normalized to β-actin from the same blot. The intensity of the control protein band was set to 1.
Figure Legend Snippet: A-E . The relative intensities of protein bands (mean ± SE) are shown in bar graphs for TLR4, connexin 43 (Cx 43), Na + /K + ATPase, GLT-1, GLAST-1. Intensity were quantified using the ImageLab software in the linear exposure range and normalized to β-actin from the same blot. The intensity of the control protein band was set to 1.

Techniques Used: Software

7) Product Images from "A new plant-specific syntaxin-6 protein may define an intracytoplasmic route for begomoviruses"

Article Title: A new plant-specific syntaxin-6 protein may define an intracytoplasmic route for begomoviruses

Journal: bioRxiv

doi: 10.1101/2020.01.10.901496

NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band. A.U. denotes arbitrary units.
Figure Legend Snippet: NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band. A.U. denotes arbitrary units.

Techniques Used: In Vivo, Bimolecular Fluorescence Complementation Assay, Expressing, Confocal Microscopy, Staining, Construct, Amplification, Immunoprecipitation, Co-Immunoprecipitation Assay, Software

8) Product Images from "Comparative Proteomic and Metabolomic Analysis of Human Osteoblasts, Differentiated from Dental Pulp Stem Cells, Hinted Crucial Signaling Pathways Promoting Osteogenesis"

Article Title: Comparative Proteomic and Metabolomic Analysis of Human Osteoblasts, Differentiated from Dental Pulp Stem Cells, Hinted Crucial Signaling Pathways Promoting Osteogenesis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22157908

Confirmation of several differentially abundant proteins by Western blotting. Densitometric analysis of protein abundance was performed using ImageLab software. Values represent the means of three biological replicates. Error bars denote the standard deviations, * indicate p ≤ 0.05. Arrows indicate densitometrically analyzed major protein bands: DSP a likely cleavage product of DSPP, BMP-2 mature protein, and functional IP3R tetramer. Abbreviations: DSP (dentin sialoprotein), DMP-1 (dentin matrix protein 1), BMP-2 (bone morphogenetic protein 2), IP3R (inositol 1,4,5-trisphosphate receptor), Col6A1 (collagen 6 alpha 1 chain), GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
Figure Legend Snippet: Confirmation of several differentially abundant proteins by Western blotting. Densitometric analysis of protein abundance was performed using ImageLab software. Values represent the means of three biological replicates. Error bars denote the standard deviations, * indicate p ≤ 0.05. Arrows indicate densitometrically analyzed major protein bands: DSP a likely cleavage product of DSPP, BMP-2 mature protein, and functional IP3R tetramer. Abbreviations: DSP (dentin sialoprotein), DMP-1 (dentin matrix protein 1), BMP-2 (bone morphogenetic protein 2), IP3R (inositol 1,4,5-trisphosphate receptor), Col6A1 (collagen 6 alpha 1 chain), GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

Techniques Used: Western Blot, Software, Functional Assay

9) Product Images from "Epidermal Growth Factor single mutants highlighted by homologs cross-conservation approach differentially affect the EGF Receptor downstream pathway"

Article Title: Epidermal Growth Factor single mutants highlighted by homologs cross-conservation approach differentially affect the EGF Receptor downstream pathway

Journal: bioRxiv

doi: 10.1101/677393

EGF variants D46T and K48T affect the EGFR downstream signaling pathway. (A) Western blot analysis of EGFR-regulated downstream gene expression of EGF variant-treated A431 cancer cells. Expression of Epidermal Growth Factor Receptor (EGFR), Phospholipase-Cγ1 (PLCγ1) and PKCζ protein in A431 cancer cell line after treatment with 100 nM WT, EGF variant D46T or variant K48T. Samples were collected on Day 3 after treatment (two duplicates). Samples were incubated with Goat Anti-Rabbit IgG StarBright Blue 700 at a 1:2000 dilution and Anti-Tubulin hFAB™ Rhodamine Antibody as a loading control at a 1:3,000 dilution for 3 hrs and washed with Blocking Buffer and Milli-Q H 2 O (22 μm filtration). Immunoreactive fluorescent labeled samples were visualized and analyzed with ImageLab Software. (B) A schematic representation of one of the EGF-EGFR-mediated signaling pathways that may be initiated in the A431 epidermoid cancer cell line. Arrows indicate the positive action of downstream gene expression, whereas arrows with flat tips indicate inhibition of gene expression. The “P+” symbol represents phosphorylation of downstream-regulated proteins. The dashed line represents the potential cellular effect regulated by altering gene expression levels involved in the depicted pathway.
Figure Legend Snippet: EGF variants D46T and K48T affect the EGFR downstream signaling pathway. (A) Western blot analysis of EGFR-regulated downstream gene expression of EGF variant-treated A431 cancer cells. Expression of Epidermal Growth Factor Receptor (EGFR), Phospholipase-Cγ1 (PLCγ1) and PKCζ protein in A431 cancer cell line after treatment with 100 nM WT, EGF variant D46T or variant K48T. Samples were collected on Day 3 after treatment (two duplicates). Samples were incubated with Goat Anti-Rabbit IgG StarBright Blue 700 at a 1:2000 dilution and Anti-Tubulin hFAB™ Rhodamine Antibody as a loading control at a 1:3,000 dilution for 3 hrs and washed with Blocking Buffer and Milli-Q H 2 O (22 μm filtration). Immunoreactive fluorescent labeled samples were visualized and analyzed with ImageLab Software. (B) A schematic representation of one of the EGF-EGFR-mediated signaling pathways that may be initiated in the A431 epidermoid cancer cell line. Arrows indicate the positive action of downstream gene expression, whereas arrows with flat tips indicate inhibition of gene expression. The “P+” symbol represents phosphorylation of downstream-regulated proteins. The dashed line represents the potential cellular effect regulated by altering gene expression levels involved in the depicted pathway.

Techniques Used: Western Blot, Expressing, Variant Assay, Incubation, Blocking Assay, Filtration, Labeling, Software, Inhibition

10) Product Images from "Surfactant protein C mutation links postnatal type 2 cell dysfunction to adult disease"

Article Title: Surfactant protein C mutation links postnatal type 2 cell dysfunction to adult disease

Journal: JCI Insight

doi: 10.1172/jci.insight.142501

Accumulation of proSP-C in P4 LQ/LQ AT2 cells. ( A ) Western blot analyses of 20 μg of AT2 cell lysates separated by SDS-PAGE. Gel was stained after transfer with Coomassie-based instant blue to assess protein loading. ( B ) Normalization of proSP-C levels in A to total protein using Bio-Rad ImageLab software. ( C ) Relative Sftpc mRNA levels in isolated AT2 cells obtained by quantitative PCR of 20 ng of cDNA. Data were normalized to Ppia expression. Cycle threshold for Ppia was constant in all genotypes and across developmental time points ( Supplemental Figure 5D ). RQ, relative quantitation. For B and C , ** P
Figure Legend Snippet: Accumulation of proSP-C in P4 LQ/LQ AT2 cells. ( A ) Western blot analyses of 20 μg of AT2 cell lysates separated by SDS-PAGE. Gel was stained after transfer with Coomassie-based instant blue to assess protein loading. ( B ) Normalization of proSP-C levels in A to total protein using Bio-Rad ImageLab software. ( C ) Relative Sftpc mRNA levels in isolated AT2 cells obtained by quantitative PCR of 20 ng of cDNA. Data were normalized to Ppia expression. Cycle threshold for Ppia was constant in all genotypes and across developmental time points ( Supplemental Figure 5D ). RQ, relative quantitation. For B and C , ** P

Techniques Used: Western Blot, SDS Page, Staining, Software, Isolation, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay

11) Product Images from "RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit"

Article Title: RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit

Journal: bioRxiv

doi: 10.1101/2020.10.07.329334

RIOK2 phosphorylation at Ser483 is required for efficient maturation of pre-40S particles. (A) MTS assays were performed on Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines at the indicated time points. ODs at 490 nm were measured using Spectramax. (B) Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines were incubated with 1 µM puromycin for the indicated times. Levels of puromycin-labelled peptides were monitored by WB using anti-puromycin antibodies. WB signals were quantified using ImageLab software and expressed as arbitrary units (a.u.). (C), (D) , Total cellular RNAs were extracted from control (Ctl), RIOK2 S483A (1 to 3) or RIOK2 S483D eHAP1 cell lines. Accumulation levels of pre-rRNAs and mature rRNAs were analyzed by NB and quantified as in Fig. 1 , n=3. (E) FISH experiments performed on RIOK2 WT (Ctl) and RIOK2 S483A eHAP1 cell lines. Pre-rRNAs were detected using a Cy5-labeled 5’-ITS1 probe. Cells were stained with DAPI to visualize nuclei, and images were captured in identical conditions. (F) Nucleolar, nuclear and cytoplasmic fluorescence signals were quantified using ImageJ software. Graph representations show fold changes in RIOK2 S483A relative to RIOK2 WT cell line (n=100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, *: P≤0.05, One-tailed Mann Whitney test).
Figure Legend Snippet: RIOK2 phosphorylation at Ser483 is required for efficient maturation of pre-40S particles. (A) MTS assays were performed on Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines at the indicated time points. ODs at 490 nm were measured using Spectramax. (B) Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines were incubated with 1 µM puromycin for the indicated times. Levels of puromycin-labelled peptides were monitored by WB using anti-puromycin antibodies. WB signals were quantified using ImageLab software and expressed as arbitrary units (a.u.). (C), (D) , Total cellular RNAs were extracted from control (Ctl), RIOK2 S483A (1 to 3) or RIOK2 S483D eHAP1 cell lines. Accumulation levels of pre-rRNAs and mature rRNAs were analyzed by NB and quantified as in Fig. 1 , n=3. (E) FISH experiments performed on RIOK2 WT (Ctl) and RIOK2 S483A eHAP1 cell lines. Pre-rRNAs were detected using a Cy5-labeled 5’-ITS1 probe. Cells were stained with DAPI to visualize nuclei, and images were captured in identical conditions. (F) Nucleolar, nuclear and cytoplasmic fluorescence signals were quantified using ImageJ software. Graph representations show fold changes in RIOK2 S483A relative to RIOK2 WT cell line (n=100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, *: P≤0.05, One-tailed Mann Whitney test).

Techniques Used: Incubation, Western Blot, Software, Fluorescence In Situ Hybridization, Labeling, Staining, Fluorescence, One-tailed Test, MANN-WHITNEY

RIOK2 phosphorylation at Ser483 facilitates its release from pre-40S particles and re-import into the nucleus (A) HEK293 cells were co-transfected with plasmids expressing HA-NOB1 and Flag-RIOK2 WT , Flag-RIOK2 S483A or Flag-RIOK2 S483D . Pre-40S particles were immunopurified via HA-NOB1 and subsequently incubated for 45 or 90 min with a buffer inducing RIOK2 release at 16°C. RIOK2 proteins in supernatant (released proteins) and retained on beads (pre-40S-bound) were analyzed by WB. Experiments with Flag-RIOK2 S483A and Flag-RIOK2 S483D were performed with different sets of Flag-RIOK2 WT as control. A representative WB experiment for Flag-RIOK2 WT is shown. (B) Quantification of released RIOK2 to bound RIOK2 at t0 WB signal ratios from (A) using ImageLab software, n=3. (C) RIOK2 WT and RIOK2 S483A eHAP1 cells were treated with Leptomycin B (LMB, 20 nM) for the indicated times. Subcellular localization of RIOK2 and NOB1 was monitored by immunofluorescence microscopy using specific antibodies. Nuclei were visualized by DAPI staining. (D), (E) , Quantification of nuclear to cytoplasmic fluorescence ratios at the indicated time points obtained in (C) for RIOK2 (D) and NOB1 (E) using ImageJ software (n=100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, 2way ANOVA tests, Bonferroni posttests).
Figure Legend Snippet: RIOK2 phosphorylation at Ser483 facilitates its release from pre-40S particles and re-import into the nucleus (A) HEK293 cells were co-transfected with plasmids expressing HA-NOB1 and Flag-RIOK2 WT , Flag-RIOK2 S483A or Flag-RIOK2 S483D . Pre-40S particles were immunopurified via HA-NOB1 and subsequently incubated for 45 or 90 min with a buffer inducing RIOK2 release at 16°C. RIOK2 proteins in supernatant (released proteins) and retained on beads (pre-40S-bound) were analyzed by WB. Experiments with Flag-RIOK2 S483A and Flag-RIOK2 S483D were performed with different sets of Flag-RIOK2 WT as control. A representative WB experiment for Flag-RIOK2 WT is shown. (B) Quantification of released RIOK2 to bound RIOK2 at t0 WB signal ratios from (A) using ImageLab software, n=3. (C) RIOK2 WT and RIOK2 S483A eHAP1 cells were treated with Leptomycin B (LMB, 20 nM) for the indicated times. Subcellular localization of RIOK2 and NOB1 was monitored by immunofluorescence microscopy using specific antibodies. Nuclei were visualized by DAPI staining. (D), (E) , Quantification of nuclear to cytoplasmic fluorescence ratios at the indicated time points obtained in (C) for RIOK2 (D) and NOB1 (E) using ImageJ software (n=100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, 2way ANOVA tests, Bonferroni posttests).

Techniques Used: Transfection, Expressing, Incubation, Western Blot, Software, Immunofluorescence, Microscopy, Staining, Fluorescence

12) Product Images from "Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23"

Article Title: Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23

Journal: Brain Pathology (Zurich, Switzerland)

doi: 10.1111/bpa.12905

Altered vGlut1 protein levels indicate developmental abnormalities in the vermis of PDYN R212W mice . (A,B) The left panel shows representative immunoblots of control, PDYN WT and PDYN R212W vermal protein lysates, stained or vGlut1 and actin. The right panel shows quantification of these immunoblots using ImageLab software. vGlut1 protein levels were increased in PDYN R212W at 2, 3 and 4 weeks compared with control mice and at 2, 4 and 8 weeks of age compared with PDYN WT mice (n = 3 per genotype). # 0.10 > p > 0.05, * P
Figure Legend Snippet: Altered vGlut1 protein levels indicate developmental abnormalities in the vermis of PDYN R212W mice . (A,B) The left panel shows representative immunoblots of control, PDYN WT and PDYN R212W vermal protein lysates, stained or vGlut1 and actin. The right panel shows quantification of these immunoblots using ImageLab software. vGlut1 protein levels were increased in PDYN R212W at 2, 3 and 4 weeks compared with control mice and at 2, 4 and 8 weeks of age compared with PDYN WT mice (n = 3 per genotype). # 0.10 > p > 0.05, * P

Techniques Used: Mouse Assay, Western Blot, Staining, Software

13) Product Images from "Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases"

Article Title: Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028017

Hydrolysis of basement membrane components in vivo . Hydrolysis of laminin (A), nidogen (B), type IV collagen (C) and perlecan (D) by BaP1 and leuc-a, as detected by Western blotting of homogenates of injected mouse gastrocnemius muscle. Groups of mice were injected in the gastrocnemius muscle with either 50 µg BaP1, 50 µg leuc-a or PBS (lane C). After 15 min, mice were sacrificed and tissue was homogenized and centrifuged to obtain the supernatant. Supernatants of muscle homogenates were separated by SDS-PAGE under reducing conditions, using a 4–15% gradient gel, and transferred to nitrocellulose membranes. Immunodetection was performed with either anti-laminin, anti-nidogen, anti-type IV collagen or anti-endorepellin antibodies, and with anti-GAPDH as loading control in tissue homogenates. Reaction was developed with a chemiluminiscent substrate. Densitometry was carried out in blots of tissue homogenates with ImageLab software; a relative quantification was performed adjusting each sample to the corresponding control.
Figure Legend Snippet: Hydrolysis of basement membrane components in vivo . Hydrolysis of laminin (A), nidogen (B), type IV collagen (C) and perlecan (D) by BaP1 and leuc-a, as detected by Western blotting of homogenates of injected mouse gastrocnemius muscle. Groups of mice were injected in the gastrocnemius muscle with either 50 µg BaP1, 50 µg leuc-a or PBS (lane C). After 15 min, mice were sacrificed and tissue was homogenized and centrifuged to obtain the supernatant. Supernatants of muscle homogenates were separated by SDS-PAGE under reducing conditions, using a 4–15% gradient gel, and transferred to nitrocellulose membranes. Immunodetection was performed with either anti-laminin, anti-nidogen, anti-type IV collagen or anti-endorepellin antibodies, and with anti-GAPDH as loading control in tissue homogenates. Reaction was developed with a chemiluminiscent substrate. Densitometry was carried out in blots of tissue homogenates with ImageLab software; a relative quantification was performed adjusting each sample to the corresponding control.

Techniques Used: In Vivo, Western Blot, Injection, Mouse Assay, SDS Page, Immunodetection, Software

14) Product Images from "Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models"

Article Title: Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.202013144

Combinations of mAbs and EGFR‐specific TKIs downregulate in vitro several receptors for survival factors and effectively block the ERK and AKT pathways NSCLC expressing single‐site mutants of EGFR, PC9 (3 × 10 6 ) or H3255 (8 × 10 6 ), were seeded in 10‐cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 h with different EGFR‐specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies (60 min), and treatment with Clarity™ Western ECL Blotting Substrates (Bio‐Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio‐Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane). PC9 cells (1 × 10 6 ) were seeded in 6‐well plates and treated as in (A). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with fluorescently labeled antibodies against EGFR, HER2, and HER3, and surface expression levels of each receptor were analyzed using flow cytometry. The normalized fluorescence intensity is shown as averages + SEM of four experiments. Significance was assessed using two‐way ANOVA followed by Dunnett’s multiple comparisons test. Note that non‐significant comparisons are not shown. See P ‐values in Appendix Table S2 . PC9 cells were seeded on coverslips and treated for 24 h as in (A). Cells were washed in acidic buffer, fixed in paraformaldehyde (4%), and incubated with specific primary antibodies, followed by an Alexa Fluor 555‐conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40× magnification). Scale bars, 40 μm. Source data are available online for this figure.
Figure Legend Snippet: Combinations of mAbs and EGFR‐specific TKIs downregulate in vitro several receptors for survival factors and effectively block the ERK and AKT pathways NSCLC expressing single‐site mutants of EGFR, PC9 (3 × 10 6 ) or H3255 (8 × 10 6 ), were seeded in 10‐cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 h with different EGFR‐specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies (60 min), and treatment with Clarity™ Western ECL Blotting Substrates (Bio‐Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio‐Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane). PC9 cells (1 × 10 6 ) were seeded in 6‐well plates and treated as in (A). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with fluorescently labeled antibodies against EGFR, HER2, and HER3, and surface expression levels of each receptor were analyzed using flow cytometry. The normalized fluorescence intensity is shown as averages + SEM of four experiments. Significance was assessed using two‐way ANOVA followed by Dunnett’s multiple comparisons test. Note that non‐significant comparisons are not shown. See P ‐values in Appendix Table S2 . PC9 cells were seeded on coverslips and treated for 24 h as in (A). Cells were washed in acidic buffer, fixed in paraformaldehyde (4%), and incubated with specific primary antibodies, followed by an Alexa Fluor 555‐conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40× magnification). Scale bars, 40 μm. Source data are available online for this figure.

Techniques Used: In Vitro, Blocking Assay, Expressing, Nucleic Acid Electrophoresis, Incubation, Western Blot, Imaging, Software, Labeling, Flow Cytometry, Fluorescence, Staining, Microscopy

15) Product Images from "Antidiabetic activities of Bolanthus spergulifolius (Caryophyllaceae) extracts on insulin-resistant 3T3-L1 adipocytes"

Article Title: Antidiabetic activities of Bolanthus spergulifolius (Caryophyllaceae) extracts on insulin-resistant 3T3-L1 adipocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0252707

qRT-PCR, Western-blot, and immunofluorescence staining showing the effect of B . spergulifolius extracts on translocation and expression of Glut-4 in 3T3-L1 adipocytes. The expression of Glucose transporter-4 (Glut-4) gene, protein and intra-cellular was quantified by qRT-PCR, Western-blot and immunofluorescence staining in preadipocytes, mature adipocytes without B . spergulifolius extracts and adipocytes treated with IC 50 doses of MeOH, EA and Aqueous of B . spergulifolius extracts for 24 h. A. Glut-4 gene expression was detected using qRT-PCR at the mRNA level. Gapdh gene was used for normalization. The relative fold change analysis was conducted using the ΔΔ C T method. qRT-PCR was analysed using ΔΔ C T from three independent experiments. Data are expressed as the relative changing of the ΔΔ C T and presented as the means ± SD. B. Representative image showing Glut-4 protein (55 kDa) levels in 3T3-L1 cells by Western-blot (20 μg/lane). C. The relative intensities of Glut-4 protein band were measured as densitometrically. Beta-Actin (β-Actin) protein was used for normalization. Data were analysed using the ImageLab software. Densitometric analysis data was presented as the relative changing of the band intensities and the means ± SD from three independent experiments. D. The microscopic image at 10X magnification showing Glut-4 expression levels and nuclear staining in 3T3-L1 cells by immunofluorescence staining. Merged images obtained using anti-Glut-4 antibody and diamido-2-phenylindole dihydrochloride (DAPI) (Scale bar: 100 μm) E . Three coverslips for each cell group were evaluated. Six distinct-randomly chosen regions in each coverslip were pohotographed using a microscope and relative intensities of immunofluorescence staining with anti-Glut4 antibody were quantified using ImageJ software. The immunofluorescence staining analysis data are presented as the relative changing of the fluorescein signal intensities and the means ± SD. To analyze statistical was used Student’s t-test (two-tailed). *p
Figure Legend Snippet: qRT-PCR, Western-blot, and immunofluorescence staining showing the effect of B . spergulifolius extracts on translocation and expression of Glut-4 in 3T3-L1 adipocytes. The expression of Glucose transporter-4 (Glut-4) gene, protein and intra-cellular was quantified by qRT-PCR, Western-blot and immunofluorescence staining in preadipocytes, mature adipocytes without B . spergulifolius extracts and adipocytes treated with IC 50 doses of MeOH, EA and Aqueous of B . spergulifolius extracts for 24 h. A. Glut-4 gene expression was detected using qRT-PCR at the mRNA level. Gapdh gene was used for normalization. The relative fold change analysis was conducted using the ΔΔ C T method. qRT-PCR was analysed using ΔΔ C T from three independent experiments. Data are expressed as the relative changing of the ΔΔ C T and presented as the means ± SD. B. Representative image showing Glut-4 protein (55 kDa) levels in 3T3-L1 cells by Western-blot (20 μg/lane). C. The relative intensities of Glut-4 protein band were measured as densitometrically. Beta-Actin (β-Actin) protein was used for normalization. Data were analysed using the ImageLab software. Densitometric analysis data was presented as the relative changing of the band intensities and the means ± SD from three independent experiments. D. The microscopic image at 10X magnification showing Glut-4 expression levels and nuclear staining in 3T3-L1 cells by immunofluorescence staining. Merged images obtained using anti-Glut-4 antibody and diamido-2-phenylindole dihydrochloride (DAPI) (Scale bar: 100 μm) E . Three coverslips for each cell group were evaluated. Six distinct-randomly chosen regions in each coverslip were pohotographed using a microscope and relative intensities of immunofluorescence staining with anti-Glut4 antibody were quantified using ImageJ software. The immunofluorescence staining analysis data are presented as the relative changing of the fluorescein signal intensities and the means ± SD. To analyze statistical was used Student’s t-test (two-tailed). *p

Techniques Used: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Translocation Assay, Expressing, Software, Microscopy, Two Tailed Test

16) Product Images from "A new plant-specific syntaxin-6 protein may define an intracytoplasmic route for begomoviruses"

Article Title: A new plant-specific syntaxin-6 protein may define an intracytoplasmic route for begomoviruses

Journal: bioRxiv

doi: 10.1101/2020.01.10.901496

NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band. A.U. denotes arbitrary units.
Figure Legend Snippet: NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band. A.U. denotes arbitrary units.

Techniques Used: In Vivo, Bimolecular Fluorescence Complementation Assay, Expressing, Confocal Microscopy, Staining, Construct, Amplification, Immunoprecipitation, Co-Immunoprecipitation Assay, Software

17) Product Images from "Fibrinogen-like protein 2 aggravates nonalcoholic steatohepatitis via interaction with TLR4, eliciting inflammation in macrophages and inducing hepatic lipid metabolism disorder"

Article Title: Fibrinogen-like protein 2 aggravates nonalcoholic steatohepatitis via interaction with TLR4, eliciting inflammation in macrophages and inducing hepatic lipid metabolism disorder

Journal: Theranostics

doi: 10.7150/thno.44297

Fgl2 disruption inhibited activation of the NLRP3 inflammasome in NASH. Total protein was obtained from liver tissues of MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls. NLRP3, pro-caspase-1, cleaved caspase-1 (caspase-1 p10), pro-IL-1β, mature IL-1β and IL-18 were analyzed by western blotting (A). BMDMs from WT and fgl2-/- mice were stimulated with LPS or FFA and tested for inflammasomes by western blotting (B). Image density was quantified using ImageLab software. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P
Figure Legend Snippet: Fgl2 disruption inhibited activation of the NLRP3 inflammasome in NASH. Total protein was obtained from liver tissues of MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls. NLRP3, pro-caspase-1, cleaved caspase-1 (caspase-1 p10), pro-IL-1β, mature IL-1β and IL-18 were analyzed by western blotting (A). BMDMs from WT and fgl2-/- mice were stimulated with LPS or FFA and tested for inflammasomes by western blotting (B). Image density was quantified using ImageLab software. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P

Techniques Used: Activation Assay, Mouse Assay, Western Blot, Software

Fgl2 disruption suppressed activation of the NF-κB and p38-MAPK signaling pathways in NASH. For in vivo examination, the total protein was obtained from liver tissues of MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls. NF-κB-p65, p38-MAPK and their phosphorylated forms were analyzed by western blotting (n=6) (A). In vitro , BMDMs from WT or fgl2-/- mice were stimulated with LPS or FFA and tested for the normal and phosphorylated levels of NF-κB and p38-MAPK by western blotting (B). Image density was quantified using ImageLab software. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P
Figure Legend Snippet: Fgl2 disruption suppressed activation of the NF-κB and p38-MAPK signaling pathways in NASH. For in vivo examination, the total protein was obtained from liver tissues of MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls. NF-κB-p65, p38-MAPK and their phosphorylated forms were analyzed by western blotting (n=6) (A). In vitro , BMDMs from WT or fgl2-/- mice were stimulated with LPS or FFA and tested for the normal and phosphorylated levels of NF-κB and p38-MAPK by western blotting (B). Image density was quantified using ImageLab software. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P

Techniques Used: Activation Assay, In Vivo, Mouse Assay, Western Blot, In Vitro, Software

18) Product Images from "Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation"

Article Title: Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation

Journal: Journal of Molecular Biology

doi: 10.1016/j.jmb.2020.166795

The cyclin A N-terminal cyclin box exploits an extended surface to interact with p27 and SKP2. (a) p27 association with CDK2-cyclin A can drive the formation of an octameric (CDK2-cyclin A) 2 -SKP1-SKP2-CKS1-p27 complex. p27 was first phosphorylated with an excess of CDK2-cyclin A and the ternary phosphorylated p27-CDK2-cyclin A complex was then purified by SEC before being incubated with equimolar SKP1-SKP2 and an excess of CKS1. This hexameric complex purified by SEC (blue trace) was then incubated with either SBP-tagged CDK2-cyclinA (orange trace) or SBP-CDK2-cyclinAmut5 (green trace). The SBP tag distinguishes these CDK2-cyclin A complexes by SEC from CDK2-cyclin A bound to p27. SBP-tagged CDK2-cyclin A forms an octameric complex but SBP-tagged CDK2-cyclin Amut 5 does not (compare the orange and blue traces, complex elution positions indicated below the chromatograms). sbpCDK2 and cyclin A are identified by stars in the relevant gels. (b) The SKP2 CAIM recruits CDK-cyclin A to affect p27 phosphorylation. An intact cyclin A-SKP2 interaction recruits a catalytic CDK-cyclin module to phosphorylate p27 within a SKP1-SKP2-CDK2-cyclin A-CKS1-p27 complex. WT substrate, CDK2 D127A -cyclin A-p27-SKP1-SKP2-CKS1; 6A substrate, CDK2 D127A -cyclin A-p27-SKP1-SKP2_6A-CKS1. p27 was detected by western blot analysis using an anti phospho-p27 antibody, lower panel, representative time-course. Band intensity was revealed using ECL reagent and quantified using background adjusted total band volume with ImageLab software (BioRad). Graph is representative of assays carried out with three biological replicates of catalytically active CDK2-cyclin A and CDK2-cyclin E as control. (c) p27 and SKP2 binding to cyclin A. Cyclin A structure, highlighted in yellow, the p27 binding surface ((L255, Q290, R293, M294, L297 identified from PDB 1JSU), in red the SSMS site that binds to SKP2 (S244, S245, M246, S247) and in magenta the RXL recruitment site identified by residues I213 and W217. L297, mutation of which blocks both p27 and SKP2 binding, is colored orange. Dotted red arrow, the proposed SKP2 binding path. S. cerevisiae CLN2 shares the docking site that recognizes the RXL motif (the “HP region” 38 with cyclin A but has an adjacent surface, defined by mutations R109, L112 and R113 that recognizes LP-motif containing substrates. These three sites form a continuous surface. Cyclin A M246 is equivalent to R109 in the CLN2 modelled structure. CLN1 model, highlighted in red, residues R109, L112, R113. The CLN2 structure was generated by implementing MODELLER 60 in HHpred 61 and then superimposed on the structure of cyclin A. (d) Proposed roles for the cyclin A-SKP2 interaction in regulating the cell cycle. The SKP2-cyclin A interaction promotes the incorporation of CDK-cyclin A complexes into SCF SKP2 (“building”). Incorporation of p27 into SKP1-SKP2-CDK1/2-cyclin A/B/E-CKS1 complexes generates complexes in which the CAIM can recruit a catalytic CDK1/2-cyclin A (“recruiting”). The cyclin A binding site on SKP2 overlaps with SKP2 binding sites for pRB and Cdh1 and so may alter the effective SKP2 concentration for alternative SKP2 regulators (“sequestering”).
Figure Legend Snippet: The cyclin A N-terminal cyclin box exploits an extended surface to interact with p27 and SKP2. (a) p27 association with CDK2-cyclin A can drive the formation of an octameric (CDK2-cyclin A) 2 -SKP1-SKP2-CKS1-p27 complex. p27 was first phosphorylated with an excess of CDK2-cyclin A and the ternary phosphorylated p27-CDK2-cyclin A complex was then purified by SEC before being incubated with equimolar SKP1-SKP2 and an excess of CKS1. This hexameric complex purified by SEC (blue trace) was then incubated with either SBP-tagged CDK2-cyclinA (orange trace) or SBP-CDK2-cyclinAmut5 (green trace). The SBP tag distinguishes these CDK2-cyclin A complexes by SEC from CDK2-cyclin A bound to p27. SBP-tagged CDK2-cyclin A forms an octameric complex but SBP-tagged CDK2-cyclin Amut 5 does not (compare the orange and blue traces, complex elution positions indicated below the chromatograms). sbpCDK2 and cyclin A are identified by stars in the relevant gels. (b) The SKP2 CAIM recruits CDK-cyclin A to affect p27 phosphorylation. An intact cyclin A-SKP2 interaction recruits a catalytic CDK-cyclin module to phosphorylate p27 within a SKP1-SKP2-CDK2-cyclin A-CKS1-p27 complex. WT substrate, CDK2 D127A -cyclin A-p27-SKP1-SKP2-CKS1; 6A substrate, CDK2 D127A -cyclin A-p27-SKP1-SKP2_6A-CKS1. p27 was detected by western blot analysis using an anti phospho-p27 antibody, lower panel, representative time-course. Band intensity was revealed using ECL reagent and quantified using background adjusted total band volume with ImageLab software (BioRad). Graph is representative of assays carried out with three biological replicates of catalytically active CDK2-cyclin A and CDK2-cyclin E as control. (c) p27 and SKP2 binding to cyclin A. Cyclin A structure, highlighted in yellow, the p27 binding surface ((L255, Q290, R293, M294, L297 identified from PDB 1JSU), in red the SSMS site that binds to SKP2 (S244, S245, M246, S247) and in magenta the RXL recruitment site identified by residues I213 and W217. L297, mutation of which blocks both p27 and SKP2 binding, is colored orange. Dotted red arrow, the proposed SKP2 binding path. S. cerevisiae CLN2 shares the docking site that recognizes the RXL motif (the “HP region” 38 with cyclin A but has an adjacent surface, defined by mutations R109, L112 and R113 that recognizes LP-motif containing substrates. These three sites form a continuous surface. Cyclin A M246 is equivalent to R109 in the CLN2 modelled structure. CLN1 model, highlighted in red, residues R109, L112, R113. The CLN2 structure was generated by implementing MODELLER 60 in HHpred 61 and then superimposed on the structure of cyclin A. (d) Proposed roles for the cyclin A-SKP2 interaction in regulating the cell cycle. The SKP2-cyclin A interaction promotes the incorporation of CDK-cyclin A complexes into SCF SKP2 (“building”). Incorporation of p27 into SKP1-SKP2-CDK1/2-cyclin A/B/E-CKS1 complexes generates complexes in which the CAIM can recruit a catalytic CDK1/2-cyclin A (“recruiting”). The cyclin A binding site on SKP2 overlaps with SKP2 binding sites for pRB and Cdh1 and so may alter the effective SKP2 concentration for alternative SKP2 regulators (“sequestering”).

Techniques Used: Purification, Incubation, Western Blot, Software, Binding Assay, Mutagenesis, Generated, Concentration Assay

19) Product Images from "Evaluation of oxidative stress effects on different macromolecules in adult growth hormone deficiency"

Article Title: Evaluation of oxidative stress effects on different macromolecules in adult growth hormone deficiency

Journal: PLoS ONE

doi: 10.1371/journal.pone.0236357

Western Blot and densitometric analysis of serum nitrotryptofan (NT) and transferrin (Tfr) levels in the three groups. (A) Western blot analysis. Blots were acquired with a Chemidoc XPS and the images were analyzed with the ImageLab software. Transferrin was used as an internal and normalization control. (B) Densitometric analysis from all the data analyzed. The relative densitometric expression of each patient was normalized with transferrin expression. Data are expressed as fold/increase (mean±SEM). Abbreviations: t-GHD: total GHD; p-GHD: partial GHD; ctrl: control subjects.
Figure Legend Snippet: Western Blot and densitometric analysis of serum nitrotryptofan (NT) and transferrin (Tfr) levels in the three groups. (A) Western blot analysis. Blots were acquired with a Chemidoc XPS and the images were analyzed with the ImageLab software. Transferrin was used as an internal and normalization control. (B) Densitometric analysis from all the data analyzed. The relative densitometric expression of each patient was normalized with transferrin expression. Data are expressed as fold/increase (mean±SEM). Abbreviations: t-GHD: total GHD; p-GHD: partial GHD; ctrl: control subjects.

Techniques Used: Western Blot, Software, Expressing

20) Product Images from "Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models"

Article Title: Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22126279

Sin3, HDAC1 and HIRA expression during a recurrent HSV-1 infection in vitro. Representative immunoblots showing Sin3 ( a ), HDAC1 ( b ), and HIRA ( c ) protein levels in lysates from Mock- and HSV-1-infected neuronal cultures harvested 24 h p.i. or 24 h p.r. Actin staining was used as a loading control. Densitometric analyses were performed with ImageLab software and normalized to actin expression. The values represent the normalized fold-changes in protein levels from 24 h p.r. CTRL or HSV-1 samples with respect to 24 h p.i. CTRL. Data are mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Sin3, HDAC1 and HIRA expression during a recurrent HSV-1 infection in vitro. Representative immunoblots showing Sin3 ( a ), HDAC1 ( b ), and HIRA ( c ) protein levels in lysates from Mock- and HSV-1-infected neuronal cultures harvested 24 h p.i. or 24 h p.r. Actin staining was used as a loading control. Densitometric analyses were performed with ImageLab software and normalized to actin expression. The values represent the normalized fold-changes in protein levels from 24 h p.r. CTRL or HSV-1 samples with respect to 24 h p.i. CTRL. Data are mean ± SEM of three independent experiments. * p

Techniques Used: Expressing, Infection, In Vitro, Western Blot, Staining, Software

Recurrent HSV-1 infection induces an increase in Sin3 and HDAC1 protein expression. Representative WBs showing Sin3 ( a ) and HDAC1 ( b ) protein levels in the whole lysate from the cortices of 4 d.p.i., Post 3TS, Pre and Post 7TS mice. Densitometric analyses were performed with ImageLab software and normalized to H3 or tubulin expression. The values represent the normalized fold-changes in protein levels from HSV1-M with respect to CTRL-M. Data are mean ± SEM. * p
Figure Legend Snippet: Recurrent HSV-1 infection induces an increase in Sin3 and HDAC1 protein expression. Representative WBs showing Sin3 ( a ) and HDAC1 ( b ) protein levels in the whole lysate from the cortices of 4 d.p.i., Post 3TS, Pre and Post 7TS mice. Densitometric analyses were performed with ImageLab software and normalized to H3 or tubulin expression. The values represent the normalized fold-changes in protein levels from HSV1-M with respect to CTRL-M. Data are mean ± SEM. * p

Techniques Used: Infection, Expressing, Mouse Assay, Software

HIRA protein expression in the cortex of recurrent HSV-1 infection mouse model. Representative WBs of HIRA protein expression 4 d.p.i., 24 h after the 3rd and the 7th TS (Post 3TS and Post 7TS respectively), or 24 h before the 7th TS (Pre 7TS). Densitometric analyses were performed with ImageLab software and normalized to actin expression level. The values represent the normalized fold-changes in protein levels from HSV1-M with respect to CTRL-M (bar graph on the left). Data are mean ± SEM. *** p
Figure Legend Snippet: HIRA protein expression in the cortex of recurrent HSV-1 infection mouse model. Representative WBs of HIRA protein expression 4 d.p.i., 24 h after the 3rd and the 7th TS (Post 3TS and Post 7TS respectively), or 24 h before the 7th TS (Pre 7TS). Densitometric analyses were performed with ImageLab software and normalized to actin expression level. The values represent the normalized fold-changes in protein levels from HSV1-M with respect to CTRL-M (bar graph on the left). Data are mean ± SEM. *** p

Techniques Used: Expressing, Infection, Software

Schematic representation of the recurrent HSV-1 infection and modulation of histone acetylation levels in vivo. ( a ) Timeline showing HSV-1 infection, TS-induced virus reactivations, and chosen experimental time-points over the mouse life. The HSV-1 inoculation (primary infection) was performed by snout abrasion (first red arrow). TS induces viral reactivations are indicated by red arrows. The dashed line indicates the establishment of the latent infection. Mice were sacrificed and analyzed at the following time points (blue dots) indicated by the yellow boxes: 4 d.p.i., following the 3rd (at 6 months of age, Post 3TS) and the 7th TS (13 months of age, Post 7TS) or 24 h just before the 7th TS (Pre 7TS). Representative WBs showing H4K16 ( b ) and H3K56 ( c ) acetylation levels in the whole lysate from the cortices of 4 d.p.i., Post 3TS, Pre and Post 7TS mice. Densitometric analyses were performed with ImageLab software and normalized to H3 or tubulin expression. The values represent the normalized fold-changes in protein levels from HSV1-M with respect to CTRL-M. Data are mean ± SEM. * p
Figure Legend Snippet: Schematic representation of the recurrent HSV-1 infection and modulation of histone acetylation levels in vivo. ( a ) Timeline showing HSV-1 infection, TS-induced virus reactivations, and chosen experimental time-points over the mouse life. The HSV-1 inoculation (primary infection) was performed by snout abrasion (first red arrow). TS induces viral reactivations are indicated by red arrows. The dashed line indicates the establishment of the latent infection. Mice were sacrificed and analyzed at the following time points (blue dots) indicated by the yellow boxes: 4 d.p.i., following the 3rd (at 6 months of age, Post 3TS) and the 7th TS (13 months of age, Post 7TS) or 24 h just before the 7th TS (Pre 7TS). Representative WBs showing H4K16 ( b ) and H3K56 ( c ) acetylation levels in the whole lysate from the cortices of 4 d.p.i., Post 3TS, Pre and Post 7TS mice. Densitometric analyses were performed with ImageLab software and normalized to H3 or tubulin expression. The values represent the normalized fold-changes in protein levels from HSV1-M with respect to CTRL-M. Data are mean ± SEM. * p

Techniques Used: Infection, In Vivo, Mouse Assay, Software, Expressing

21) Product Images from "Mesenchymal stem cells improve mouse non-heart-beating liver graft survival by inhibiting Kupffer cell apoptosis via TLR4-ERK1/2-Fas/FasL-caspase3 pathway regulation"

Article Title: Mesenchymal stem cells improve mouse non-heart-beating liver graft survival by inhibiting Kupffer cell apoptosis via TLR4-ERK1/2-Fas/FasL-caspase3 pathway regulation

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-016-0416-y

Mesenchymal stem cells ( MSCs ) inhibit Kupffer cell apoptosis via the PGE2-TLR4-ERK1/2-Fas/FasL-caspase3 pathway. Murine-derived RAW264.7 cells were used to represent Kupffer cells; 5 × 10 5 macrophages per well were seeded in a six-well plate, and 10 5 MSCs were seeded in a 0.4-μm polycarbonate membrane insert Transwell in medium containing or lacking NS-398 (5 μM). After 24 h, the medium was changed once to remove non-adherent dead cells. After 2 h of incubation at 37 °C without O 2 , the macrophages were transferred into co-culture with the Transwells containing MSCs, and 0, 100, 200, 400, or 800 μM hydrogen peroxide ( H 2 O 2 ) was added to specific wells. Then, the co-cultured cells were incubated at 37 °C with 5 % CO 2 for 6 h to mimic the process of IRI during NHB liver transplantation in vivo. a RAW264.7 cells were stained with F4/80 antibody, a surface marker of Kupffer cells, and analysed by flow cytometry. Representative images from one experiment are shown. Blue shading and red shading indicate immunofluorescence intensity of cells for the isotype and F4/80 antibodies, respectively. b In vitro co-culture system. After H 2 O 2 stimulation for 6 hours, apoptosis of Kupffer cells was detected by Annexin V/PI staining. Representative images from one experiment out of three are shown. c Summarized results are presented as mean ± SEM ( N = 3 for each group). d After H 2 O 2 stimulation for 6 hours, expression of TLR2, TLR4, ERK1/2, p-ERK1/2, Fas, FasL, cleaved caspase3, and β-actin from Kupffer cells was determined by Western blotting analysis. Representative images from one experiment are shown. e Grey value of each band was calculated by ImageLab software. Representing relative expression of target proteins, the grey value ratio compared with β-actin was analysed and shown as mean ± SEM ( N = 3 for each group). * P
Figure Legend Snippet: Mesenchymal stem cells ( MSCs ) inhibit Kupffer cell apoptosis via the PGE2-TLR4-ERK1/2-Fas/FasL-caspase3 pathway. Murine-derived RAW264.7 cells were used to represent Kupffer cells; 5 × 10 5 macrophages per well were seeded in a six-well plate, and 10 5 MSCs were seeded in a 0.4-μm polycarbonate membrane insert Transwell in medium containing or lacking NS-398 (5 μM). After 24 h, the medium was changed once to remove non-adherent dead cells. After 2 h of incubation at 37 °C without O 2 , the macrophages were transferred into co-culture with the Transwells containing MSCs, and 0, 100, 200, 400, or 800 μM hydrogen peroxide ( H 2 O 2 ) was added to specific wells. Then, the co-cultured cells were incubated at 37 °C with 5 % CO 2 for 6 h to mimic the process of IRI during NHB liver transplantation in vivo. a RAW264.7 cells were stained with F4/80 antibody, a surface marker of Kupffer cells, and analysed by flow cytometry. Representative images from one experiment are shown. Blue shading and red shading indicate immunofluorescence intensity of cells for the isotype and F4/80 antibodies, respectively. b In vitro co-culture system. After H 2 O 2 stimulation for 6 hours, apoptosis of Kupffer cells was detected by Annexin V/PI staining. Representative images from one experiment out of three are shown. c Summarized results are presented as mean ± SEM ( N = 3 for each group). d After H 2 O 2 stimulation for 6 hours, expression of TLR2, TLR4, ERK1/2, p-ERK1/2, Fas, FasL, cleaved caspase3, and β-actin from Kupffer cells was determined by Western blotting analysis. Representative images from one experiment are shown. e Grey value of each band was calculated by ImageLab software. Representing relative expression of target proteins, the grey value ratio compared with β-actin was analysed and shown as mean ± SEM ( N = 3 for each group). * P

Techniques Used: Derivative Assay, Incubation, Co-Culture Assay, Cell Culture, Transplantation Assay, In Vivo, Staining, Marker, Flow Cytometry, Cytometry, Immunofluorescence, In Vitro, Expressing, Western Blot, Software

22) Product Images from "Helicobacter pylori binds human Annexins via Lipopolysaccharide to interfere with Toll-like Receptor 4 signaling"

Article Title: Helicobacter pylori binds human Annexins via Lipopolysaccharide to interfere with Toll-like Receptor 4 signaling

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1010326

Effect of ANXA5 binding on CagA translocation of H . pylori . A-B ) AGS cells were infected with H . pylori strain P12-GFP. H . pylori has been pre-incubated with ANXA5 before infection. A ) Shown is the optical density of the CagA P12-GFP P-Tyr band with ANXA5 pre-incubation. Quantification of CagA P-Tyr was performed using ImageLab software (Version 6.0.1, BioRad) and normalized to the stain-free gel as shown in (B). B ) Representative blots of phosphorylated CagA (P-Tyr), CagA and RecA as well as the stain-free-image are shown. Statistical analysis was performed with one-way ANOVA and Bonferroni’s post test; n = 4. * p
Figure Legend Snippet: Effect of ANXA5 binding on CagA translocation of H . pylori . A-B ) AGS cells were infected with H . pylori strain P12-GFP. H . pylori has been pre-incubated with ANXA5 before infection. A ) Shown is the optical density of the CagA P12-GFP P-Tyr band with ANXA5 pre-incubation. Quantification of CagA P-Tyr was performed using ImageLab software (Version 6.0.1, BioRad) and normalized to the stain-free gel as shown in (B). B ) Representative blots of phosphorylated CagA (P-Tyr), CagA and RecA as well as the stain-free-image are shown. Statistical analysis was performed with one-way ANOVA and Bonferroni’s post test; n = 4. * p

Techniques Used: Binding Assay, Translocation Assay, Infection, Incubation, Software, Staining

23) Product Images from "Mycobacterium tuberculosis Induced Osteoblast Dysregulation Involved in Bone Destruction in Spinal Tuberculosis"

Article Title: Mycobacterium tuberculosis Induced Osteoblast Dysregulation Involved in Bone Destruction in Spinal Tuberculosis

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2022.780272

Mce4a-ASO reduced Mycobacterium tuberculosis ( Mtb ) survival and cytokine secretion in human osteoblast cells. Osteoblast cells were incubated with Mtb in the presence or absence of Mce4a-ASO (0–10 µM) for 48 h. (A) Representative images of the colony forming unit (CFU) Petri dishes. ( a ) Mtb only. ( b ) Mtb infection with 0.1 µM Mce4a-ASO. ( c ) Mtb infection with 1 µM Mce4a-ASO. ( d ) Mtb infection with 10 µM Mce4a-ASO. (B) The number of bacterial colonies was determined using ImageLab ® software. (C) The secretion level of interleukin 6 (IL-6) was measured using a commercially available ELISA kit. (D) Osteoprotegerin produced by osteoblasts was measured using a commercially available ELISA kit according to the manufacturer’s protocol. Means without a common letter differ. p
Figure Legend Snippet: Mce4a-ASO reduced Mycobacterium tuberculosis ( Mtb ) survival and cytokine secretion in human osteoblast cells. Osteoblast cells were incubated with Mtb in the presence or absence of Mce4a-ASO (0–10 µM) for 48 h. (A) Representative images of the colony forming unit (CFU) Petri dishes. ( a ) Mtb only. ( b ) Mtb infection with 0.1 µM Mce4a-ASO. ( c ) Mtb infection with 1 µM Mce4a-ASO. ( d ) Mtb infection with 10 µM Mce4a-ASO. (B) The number of bacterial colonies was determined using ImageLab ® software. (C) The secretion level of interleukin 6 (IL-6) was measured using a commercially available ELISA kit. (D) Osteoprotegerin produced by osteoblasts was measured using a commercially available ELISA kit according to the manufacturer’s protocol. Means without a common letter differ. p

Techniques Used: Allele-specific Oligonucleotide, Incubation, Infection, Software, Enzyme-linked Immunosorbent Assay, Produced

24) Product Images from "Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency"

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency

Journal: Retrovirology

doi: 10.1186/s12977-022-00605-4

1H3 affects CLK1 and CLK2 function and expression. a Effect of compound 1H3 on SRSF2 subcellular distribution upon overexpression of CLKs in HeLa B2 cell line. Cells were transfected with indicated GFP-CLK expression vectors and 48 h post transfection treated with DMSO or 200 nM 1H3 for 24 h, fixed and processed for immunofluorescence. Cells were stained with anti-SRSF2 antibody, a marker for nuclear speckles, and nuclei stained with DAPI. Shown are the representative images of the localization patterns of SRSF2 upon overexpression of CLK1, CLK2, or CLK3. Red arrows indicate loss of nuclear speckles due to CLK overexpression in DMSO or 1H3-treated cells upon CLK3 overexpression. Yellow arrows indicate restoration of nuclear speckles in 1H3-treated cells upon CLK1 or CLK2 overexpression. Images are representative of n = 3 independent experiments. b , c Healthy donor CD4+ T cells were infected with HIV-1 89.6 then treated with DMSO, 10 µM TG003, or 300 nM 1H3 for 3 d. Cells were harvested and the effect of individual treatments on CLK1, CLK2, CLK3, or SRPK1 b protein or c mRNA expression determined. Shown on the left in b is a representative western blot and, on the right, a summary of assays. Data shown corresponds to results from n > 6 individual patient samples from 2 independent experiments. d , e 1C8 inhibits HIV-1 gene expression and alters CLK expression. CEM-HIV* cells were treated with 10 µM 1C8 or equivalent volume of DMSO. HIV-1 expression was induced by addition of DOX + prostratin for 24 h. Cells were subsequently harvested, and the lysates analyzed by western blot for d HIV-1 Gag and Env expression or e expression of CLK1, CLK2, or CLK3. Shown are the representative western blots and below is a graphical summary of the blots across n = 3 independent assays. Band intensity was quantified relative to Dox induced DMSO control and normalized to total protein load using Bio-Rad ImageLab software. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001
Figure Legend Snippet: 1H3 affects CLK1 and CLK2 function and expression. a Effect of compound 1H3 on SRSF2 subcellular distribution upon overexpression of CLKs in HeLa B2 cell line. Cells were transfected with indicated GFP-CLK expression vectors and 48 h post transfection treated with DMSO or 200 nM 1H3 for 24 h, fixed and processed for immunofluorescence. Cells were stained with anti-SRSF2 antibody, a marker for nuclear speckles, and nuclei stained with DAPI. Shown are the representative images of the localization patterns of SRSF2 upon overexpression of CLK1, CLK2, or CLK3. Red arrows indicate loss of nuclear speckles due to CLK overexpression in DMSO or 1H3-treated cells upon CLK3 overexpression. Yellow arrows indicate restoration of nuclear speckles in 1H3-treated cells upon CLK1 or CLK2 overexpression. Images are representative of n = 3 independent experiments. b , c Healthy donor CD4+ T cells were infected with HIV-1 89.6 then treated with DMSO, 10 µM TG003, or 300 nM 1H3 for 3 d. Cells were harvested and the effect of individual treatments on CLK1, CLK2, CLK3, or SRPK1 b protein or c mRNA expression determined. Shown on the left in b is a representative western blot and, on the right, a summary of assays. Data shown corresponds to results from n > 6 individual patient samples from 2 independent experiments. d , e 1C8 inhibits HIV-1 gene expression and alters CLK expression. CEM-HIV* cells were treated with 10 µM 1C8 or equivalent volume of DMSO. HIV-1 expression was induced by addition of DOX + prostratin for 24 h. Cells were subsequently harvested, and the lysates analyzed by western blot for d HIV-1 Gag and Env expression or e expression of CLK1, CLK2, or CLK3. Shown are the representative western blots and below is a graphical summary of the blots across n = 3 independent assays. Band intensity was quantified relative to Dox induced DMSO control and normalized to total protein load using Bio-Rad ImageLab software. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Techniques Used: Expressing, Over Expression, Transfection, Immunofluorescence, Staining, Marker, Infection, Western Blot, Software

Depletion of SR kinases has differential effects on HIV-1 protein levels. a Schematic of HIV-1 rtTAGagzipGFP provirus used to generate CEM-HIV* cell line. b , c CEM-HIV* cells were infected with shRNA lentivirus targeting CLK1, CLK2, CLK3, or SRPK1 and transduced cells were selected with puromycin for 72 h. Following puromycin selection, HIV-1 gene expression was induced with doxycycline (Dox, 4.5 µM) + prostratin (Pros,2.56 µM) and cells harvested for western blots after 24 h of induction. Shown are the representative western blots indicating expression levels of b the target kinase, c or HIV-1 Env, Gag, and Tat levels. Band intensity was quantified relative to Dox induced shRNA control and normalized to either total protein stain for Env and Gag blots or GAPDH for Tat blots using Bio-Rad ImageLab software. Data are indicated as mean ± SEM, n ≥ 4 independent experiments, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Dotted vertical lines on the blots represent cropping of lanes on the same representative blot to show shcontrol lanes adjacent to shRNA target depletion lanes
Figure Legend Snippet: Depletion of SR kinases has differential effects on HIV-1 protein levels. a Schematic of HIV-1 rtTAGagzipGFP provirus used to generate CEM-HIV* cell line. b , c CEM-HIV* cells were infected with shRNA lentivirus targeting CLK1, CLK2, CLK3, or SRPK1 and transduced cells were selected with puromycin for 72 h. Following puromycin selection, HIV-1 gene expression was induced with doxycycline (Dox, 4.5 µM) + prostratin (Pros,2.56 µM) and cells harvested for western blots after 24 h of induction. Shown are the representative western blots indicating expression levels of b the target kinase, c or HIV-1 Env, Gag, and Tat levels. Band intensity was quantified relative to Dox induced shRNA control and normalized to either total protein stain for Env and Gag blots or GAPDH for Tat blots using Bio-Rad ImageLab software. Data are indicated as mean ± SEM, n ≥ 4 independent experiments, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Dotted vertical lines on the blots represent cropping of lanes on the same representative blot to show shcontrol lanes adjacent to shRNA target depletion lanes

Techniques Used: Infection, shRNA, Selection, Expressing, Western Blot, Staining, Software

Activation of primary CD4+ T cells selectively alters SR kinase and SR protein levels. Primary CD4+ T cells were isolated from healthy (HIV uninfected) human donors and untreated (control) or treated with activators (anti-CD3/CD28 and IL-2). Cells were harvested at different times (24 h, 48 h, 4 d, and 6 d) with or without activation for analyses by western blots or RT-qPCR to look for changes in the expression of SR kinases and SR proteins. a Top and bottom panels on the left are the representative western blots probed for CLK1, CLK2, CLK3, and SRPK1. Top and bottom panels on the right are the quantitation of blots for at least 3 donors (except for 4 d and 6 d post-activation for CLK2, CLK3, and SRPK1 expression levels where only one donor was used). b Quantification of CLK1 and SRPK1 mRNA levels in CD4+ T cells of 3 donors by RT-qPCR assay. mRNA levels were normalized to ß2-microglobulin and mean mRNA levels were expressed relative to untreated control. c Quantitation of western blots for SR protein expression levels in untreated versus treated/activated CD4+ T cell lysates (see Additional file 1 : Fig. S4 for representative western blots) across at least 3 donors. For western blots, band intensity was quantified relative to untreated control and normalized to total protein load using Bio-Rad ImageLab software. Data are indicated as mean ± SD, n = 3 or 4 independent experiments, *p ≤ 0.05 and **p ≤ 0.01
Figure Legend Snippet: Activation of primary CD4+ T cells selectively alters SR kinase and SR protein levels. Primary CD4+ T cells were isolated from healthy (HIV uninfected) human donors and untreated (control) or treated with activators (anti-CD3/CD28 and IL-2). Cells were harvested at different times (24 h, 48 h, 4 d, and 6 d) with or without activation for analyses by western blots or RT-qPCR to look for changes in the expression of SR kinases and SR proteins. a Top and bottom panels on the left are the representative western blots probed for CLK1, CLK2, CLK3, and SRPK1. Top and bottom panels on the right are the quantitation of blots for at least 3 donors (except for 4 d and 6 d post-activation for CLK2, CLK3, and SRPK1 expression levels where only one donor was used). b Quantification of CLK1 and SRPK1 mRNA levels in CD4+ T cells of 3 donors by RT-qPCR assay. mRNA levels were normalized to ß2-microglobulin and mean mRNA levels were expressed relative to untreated control. c Quantitation of western blots for SR protein expression levels in untreated versus treated/activated CD4+ T cell lysates (see Additional file 1 : Fig. S4 for representative western blots) across at least 3 donors. For western blots, band intensity was quantified relative to untreated control and normalized to total protein load using Bio-Rad ImageLab software. Data are indicated as mean ± SD, n = 3 or 4 independent experiments, *p ≤ 0.05 and **p ≤ 0.01

Techniques Used: Activation Assay, Isolation, Western Blot, Quantitative RT-PCR, Expressing, Quantitation Assay, Software

25) Product Images from "CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis"

Article Title: CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

Journal: Scientific Reports

doi: 10.1038/s41598-017-18442-3

Setup of pMN:: fkbp-cas9 and pMN::gRNA constructs. ( A ) Cartoon representation (not to scale) of pMN:: fkbp-cas9 plasmid. The T. vaginalis drive expression of the fkbp (FKBP destabilization domain)- cas9 fusion gene. The cas9 . ( B ) Representative immunoblot analysis (inset) and quantification of replicate samples of pMN:: fkbp-cas9 expression. Samples were induced for 24 hours with 0, 0.1, 0.5 or 1 μM Shield-1 ligand before protein was collected and resolved by SDS-PAGE. Inset, upper: anti-Cas9 immunoblot (Clontech); Inset, lower: anti-TvGAPDH antibody (Cocalico Biologicals). Control sample used a pMN::EV (pMasterNEO::empty vector) transfectant. Quantitative comparison of samples utilized normalized signal of Cas9 (upper blot) to GAPDH signal (lower blot) taken from within an experiment and analyzed in parallel on separate immunoblots using a Bio-Rad Gel Doc and ImageLab software. Bar graph represents average +/− standard deviation of two independent analyses. ( C ) Cartoon representation (not to scale) of pMN:: gRNA – 360 bp of the T. vaginalis U6 5′ UTR and 37 bp of the 3′ UTR flank the gRNA. A 20 nt seed region with the first nucleotide always an adenine residue followed by 19 nt seed region used for specific targeting and the gRNA scaffold. ( D ) RT-PCR products amplifying either the gRNA sequence or the U6 snRNA (control). Total RNA was subjected to +/− reverse transcriptase then amplified by PCR using gRNA- or U6 snRNA-specific primers. Control PCRs included the pMN::EV and pMN:: gRNA .
Figure Legend Snippet: Setup of pMN:: fkbp-cas9 and pMN::gRNA constructs. ( A ) Cartoon representation (not to scale) of pMN:: fkbp-cas9 plasmid. The T. vaginalis drive expression of the fkbp (FKBP destabilization domain)- cas9 fusion gene. The cas9 . ( B ) Representative immunoblot analysis (inset) and quantification of replicate samples of pMN:: fkbp-cas9 expression. Samples were induced for 24 hours with 0, 0.1, 0.5 or 1 μM Shield-1 ligand before protein was collected and resolved by SDS-PAGE. Inset, upper: anti-Cas9 immunoblot (Clontech); Inset, lower: anti-TvGAPDH antibody (Cocalico Biologicals). Control sample used a pMN::EV (pMasterNEO::empty vector) transfectant. Quantitative comparison of samples utilized normalized signal of Cas9 (upper blot) to GAPDH signal (lower blot) taken from within an experiment and analyzed in parallel on separate immunoblots using a Bio-Rad Gel Doc and ImageLab software. Bar graph represents average +/− standard deviation of two independent analyses. ( C ) Cartoon representation (not to scale) of pMN:: gRNA – 360 bp of the T. vaginalis U6 5′ UTR and 37 bp of the 3′ UTR flank the gRNA. A 20 nt seed region with the first nucleotide always an adenine residue followed by 19 nt seed region used for specific targeting and the gRNA scaffold. ( D ) RT-PCR products amplifying either the gRNA sequence or the U6 snRNA (control). Total RNA was subjected to +/− reverse transcriptase then amplified by PCR using gRNA- or U6 snRNA-specific primers. Control PCRs included the pMN::EV and pMN:: gRNA .

Techniques Used: Construct, Plasmid Preparation, Expressing, SDS Page, Transfection, Western Blot, Software, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Polymerase Chain Reaction

CRISPR-directed KO of ferredoxin-1 and mif . ( A ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance gene flanked by the ferredoxin-1 UTRs and a plasmid containing Cas9 and Fd gRNA (pCas9-gRNA( Fd )) followed by selection for G418-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( B ) PCR analysis of G418-resistant clones for the presence of the neo resistance gene in the ferredoxin-1 locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5 A) combined with primers specific to the ferredoxin-1 locus (blue arrows in 5 A) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1665 bp and the 3′ PCR screen (middle panel) predicts a product of 1236 bp if the neo gene is present in the ferredoxin-1 locus. Lowest panel: PCR analysis of the ferredoxin-1 gene in clones. Amplification utilized primers specific to the wild-type ferredoxin-1 (yielding a 324 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( C ) Immunoblot protein analysis of the clones yielding positive PCR products for KO in Fig. 5B. Total protein extracts were resolved by SDS-PAGE and immunoblotted using anti-Ferredoxin-1 and TvGAPDH antibodies. All immunoblots contained equal amounts of the same experimental samples and were analyzed in parallel using a Bio-Rad Gel Doc and ImageLab software. ( D ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance (NeoR) gene flanked by the mif UTRs and a plasmid containing Cas9 and mif gRNAs (pCas9-gRNA( mif )), followed by selection for G418- and puromycin-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( E ) PCR analysis of G418 and puromycin-resistant clones for the presence of the neo resistance gene in the mif locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5D) combined with primers specific to the mif locus (orange arrows in 5D) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1290 bp and the 3′ PCR screen (middle panel) predicts a product of 1220 bp if the neo gene is present in the mif locus. Lowest panel: PCR analysis of the mif gene in clones. Amplification utilized primers specific to the wild-type mif gene (yielding a 500 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( F .
Figure Legend Snippet: CRISPR-directed KO of ferredoxin-1 and mif . ( A ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance gene flanked by the ferredoxin-1 UTRs and a plasmid containing Cas9 and Fd gRNA (pCas9-gRNA( Fd )) followed by selection for G418-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( B ) PCR analysis of G418-resistant clones for the presence of the neo resistance gene in the ferredoxin-1 locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5 A) combined with primers specific to the ferredoxin-1 locus (blue arrows in 5 A) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1665 bp and the 3′ PCR screen (middle panel) predicts a product of 1236 bp if the neo gene is present in the ferredoxin-1 locus. Lowest panel: PCR analysis of the ferredoxin-1 gene in clones. Amplification utilized primers specific to the wild-type ferredoxin-1 (yielding a 324 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( C ) Immunoblot protein analysis of the clones yielding positive PCR products for KO in Fig. 5B. Total protein extracts were resolved by SDS-PAGE and immunoblotted using anti-Ferredoxin-1 and TvGAPDH antibodies. All immunoblots contained equal amounts of the same experimental samples and were analyzed in parallel using a Bio-Rad Gel Doc and ImageLab software. ( D ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance (NeoR) gene flanked by the mif UTRs and a plasmid containing Cas9 and mif gRNAs (pCas9-gRNA( mif )), followed by selection for G418- and puromycin-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( E ) PCR analysis of G418 and puromycin-resistant clones for the presence of the neo resistance gene in the mif locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5D) combined with primers specific to the mif locus (orange arrows in 5D) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1290 bp and the 3′ PCR screen (middle panel) predicts a product of 1220 bp if the neo gene is present in the mif locus. Lowest panel: PCR analysis of the mif gene in clones. Amplification utilized primers specific to the wild-type mif gene (yielding a 500 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( F .

Techniques Used: CRISPR, Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Clone Assay, Amplification, Software, SDS Page, Western Blot

26) Product Images from "p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway"

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

Journal: Cell Death & Disease

doi: 10.1038/s41419-021-04224-3

Altered PCNA ubiquitination after expression of p53α or alternative isoforms. K562 cells were transfected with expression plasmids for p53α, alternative p53 isoforms [ A p53β, p53γ, B Δ40p53α, Δ133p53α and Δ160p53α] or EV in controls (ctrl). Forty-eight hours after transfection, cells were either mock- or MMC-treated (3 μM, 45 min, 3 h release), subsequently cells were lysed with either lysis buffer for protein extraction (50 mM Tris [pH7.4], 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM Sodium fluoride, 25 mM β-Glycerol phosphate, 0.1 mM Sodium vanadate, 0.2% Triton X-100, 0.3 % Nonidet P40, complete protease inhibitor [Roche]) or IP lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP40, complete protease inhibitor [Roche]) and then processed for immunoblotting using ubiquityl PCNA (Lys164, D5C7P, Cell Signaling, Massachusetts, USA) antibody, recognizing PCNA protein only when ubiquitinated at Lys164, as well as antibodies against PCNA and p53. β-Actin was used as loading control. “ub” indicates ubiquitination. Quantification of respective protein expression level was carried out using ImageLab software. Levels of PCNA mono-/polyubiquitination were corrected for PCNA and normalized to ctrl ( C , D ) which was set to 1 on each blot. Statistically significant differences among groups were calculated by Friedman test followed by Wilcoxon-signed ranks test in case of statistical significance. Representative Western Blots from cells expressing ctrl, p53α, and C-terminal isoforms ( A ) or cells expressing ctrl, p53α, and ΔN-isoforms ( B ). Heatmap of mean values from ≥ 5 independent experiments ( C ) or ≥ 4 independent experiments ( D ).
Figure Legend Snippet: Altered PCNA ubiquitination after expression of p53α or alternative isoforms. K562 cells were transfected with expression plasmids for p53α, alternative p53 isoforms [ A p53β, p53γ, B Δ40p53α, Δ133p53α and Δ160p53α] or EV in controls (ctrl). Forty-eight hours after transfection, cells were either mock- or MMC-treated (3 μM, 45 min, 3 h release), subsequently cells were lysed with either lysis buffer for protein extraction (50 mM Tris [pH7.4], 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM Sodium fluoride, 25 mM β-Glycerol phosphate, 0.1 mM Sodium vanadate, 0.2% Triton X-100, 0.3 % Nonidet P40, complete protease inhibitor [Roche]) or IP lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP40, complete protease inhibitor [Roche]) and then processed for immunoblotting using ubiquityl PCNA (Lys164, D5C7P, Cell Signaling, Massachusetts, USA) antibody, recognizing PCNA protein only when ubiquitinated at Lys164, as well as antibodies against PCNA and p53. β-Actin was used as loading control. “ub” indicates ubiquitination. Quantification of respective protein expression level was carried out using ImageLab software. Levels of PCNA mono-/polyubiquitination were corrected for PCNA and normalized to ctrl ( C , D ) which was set to 1 on each blot. Statistically significant differences among groups were calculated by Friedman test followed by Wilcoxon-signed ranks test in case of statistical significance. Representative Western Blots from cells expressing ctrl, p53α, and C-terminal isoforms ( A ) or cells expressing ctrl, p53α, and ΔN-isoforms ( B ). Heatmap of mean values from ≥ 5 independent experiments ( C ) or ≥ 4 independent experiments ( D ).

Techniques Used: Expressing, Transfection, Lysis, Protein Extraction, Protease Inhibitor, Software, Western Blot

Knockdown of endogenous p53-isoforms induces changes in fiber track lengths. U2OS cells ( A ) or HSPCs ( B ) were transfected with nonsense siRNA (sictrl), siRNA targeting all p53-isoforms (sip53) or siRNA targeting Δ133p53/Δ160p53 isoforms (siΔ133p53). A, B : 24 h after transfection, cells were sequentially incubated with CldU (20 µM) and IdU (200 µM) for 20 min. During IdU-incorporation, cells were either mock-treated or treated with 3 µM MMC. Both CldU- and IdU-tracks were measured while only IdU-tracks in ongoing forks (≥257 fibers from two independent biological experiments) are graphically presented for clarity. Knockdown of p53-isoforms was verified by immunoblot staining using anti-p53 (DO-11, MCA1704, Biorad) shown in the right panel of ( A, B ) each. Quantification of Δ133p53α relative to α-Tubulin levels was achieved by Imagelab and is indicated above the blots. The dashed frame in ( A ) marks an unspecific band stained by DO-11 in U2OS cells that had to be cut out, followed by antibody reincubation and a long exposure for 300 s to permit immunodetection of the Δ133p53α-band. Statistically significant differences between groups were calculated by Dunn’s multiple comparisons test. * P
Figure Legend Snippet: Knockdown of endogenous p53-isoforms induces changes in fiber track lengths. U2OS cells ( A ) or HSPCs ( B ) were transfected with nonsense siRNA (sictrl), siRNA targeting all p53-isoforms (sip53) or siRNA targeting Δ133p53/Δ160p53 isoforms (siΔ133p53). A, B : 24 h after transfection, cells were sequentially incubated with CldU (20 µM) and IdU (200 µM) for 20 min. During IdU-incorporation, cells were either mock-treated or treated with 3 µM MMC. Both CldU- and IdU-tracks were measured while only IdU-tracks in ongoing forks (≥257 fibers from two independent biological experiments) are graphically presented for clarity. Knockdown of p53-isoforms was verified by immunoblot staining using anti-p53 (DO-11, MCA1704, Biorad) shown in the right panel of ( A, B ) each. Quantification of Δ133p53α relative to α-Tubulin levels was achieved by Imagelab and is indicated above the blots. The dashed frame in ( A ) marks an unspecific band stained by DO-11 in U2OS cells that had to be cut out, followed by antibody reincubation and a long exposure for 300 s to permit immunodetection of the Δ133p53α-band. Statistically significant differences between groups were calculated by Dunn’s multiple comparisons test. * P

Techniques Used: Transfection, Incubation, Staining, Immunodetection

27) Product Images from "A plant-specific syntaxin-6 protein contributes to the intracytoplasmic route for begomoviruses"

Article Title: A plant-specific syntaxin-6 protein contributes to the intracytoplasmic route for begomoviruses

Journal: bioRxiv

doi: 10.1101/2020.01.10.901496

NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band, revealed in the same blot as the Co-IP band. A.U. denotes arbitrary units.
Figure Legend Snippet: NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band, revealed in the same blot as the Co-IP band. A.U. denotes arbitrary units.

Techniques Used: In Vivo, Bimolecular Fluorescence Complementation Assay, Expressing, Confocal Microscopy, Staining, Construct, Amplification, Immunoprecipitation, Co-Immunoprecipitation Assay, Software

28) Product Images from "Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases"

Article Title: Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028017

Hydrolysis of basement membrane components in vivo . Hydrolysis of laminin (A), nidogen (B), type IV collagen (C) and perlecan (D) by BaP1 and leuc-a, as detected by Western blotting of homogenates of injected mouse gastrocnemius muscle. Groups of mice were injected in the gastrocnemius muscle with either 50 µg BaP1, 50 µg leuc-a or PBS (lane C). After 15 min, mice were sacrificed and tissue was homogenized and centrifuged to obtain the supernatant. Supernatants of muscle homogenates were separated by SDS-PAGE under reducing conditions, using a 4–15% gradient gel, and transferred to nitrocellulose membranes. Immunodetection was performed with either anti-laminin, anti-nidogen, anti-type IV collagen or anti-endorepellin antibodies, and with anti-GAPDH as loading control in tissue homogenates. Reaction was developed with a chemiluminiscent substrate. Densitometry was carried out in blots of tissue homogenates with ImageLab software; a relative quantification was performed adjusting each sample to the corresponding control.
Figure Legend Snippet: Hydrolysis of basement membrane components in vivo . Hydrolysis of laminin (A), nidogen (B), type IV collagen (C) and perlecan (D) by BaP1 and leuc-a, as detected by Western blotting of homogenates of injected mouse gastrocnemius muscle. Groups of mice were injected in the gastrocnemius muscle with either 50 µg BaP1, 50 µg leuc-a or PBS (lane C). After 15 min, mice were sacrificed and tissue was homogenized and centrifuged to obtain the supernatant. Supernatants of muscle homogenates were separated by SDS-PAGE under reducing conditions, using a 4–15% gradient gel, and transferred to nitrocellulose membranes. Immunodetection was performed with either anti-laminin, anti-nidogen, anti-type IV collagen or anti-endorepellin antibodies, and with anti-GAPDH as loading control in tissue homogenates. Reaction was developed with a chemiluminiscent substrate. Densitometry was carried out in blots of tissue homogenates with ImageLab software; a relative quantification was performed adjusting each sample to the corresponding control.

Techniques Used: In Vivo, Western Blot, Injection, Mouse Assay, SDS Page, Immunodetection, Software

29) Product Images from "Handcuffing reversal is facilitated by proteases and replication initiator monomers"

Article Title: Handcuffing reversal is facilitated by proteases and replication initiator monomers

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx166

RK2 plasmid handcuff complex is formed by wild-type TrfA. Schematic representation of the experimental procedure is presented in panel ( A ) NA assay, and ( C ) ligation assay. The formation of handcuff complexes was analyzed using the following proteins: wild-type TrfA, monomeric form TrfA G254D/S267L and dimeric form TrfA S257F, as described under Materials and methods. The results of performed experiments are presented in panel ( B ) NA assay, and ( D ) ligase assay. On agarose gel free DNA and DNA multimers are marked F and M respectively. A quantified densitometry with using ChemiDoc MP System and ImageLab™ Software (BioRad) were applied for DNA multimers amount estimation. The error bars are derived from three different experiments.
Figure Legend Snippet: RK2 plasmid handcuff complex is formed by wild-type TrfA. Schematic representation of the experimental procedure is presented in panel ( A ) NA assay, and ( C ) ligation assay. The formation of handcuff complexes was analyzed using the following proteins: wild-type TrfA, monomeric form TrfA G254D/S267L and dimeric form TrfA S257F, as described under Materials and methods. The results of performed experiments are presented in panel ( B ) NA assay, and ( D ) ligase assay. On agarose gel free DNA and DNA multimers are marked F and M respectively. A quantified densitometry with using ChemiDoc MP System and ImageLab™ Software (BioRad) were applied for DNA multimers amount estimation. The error bars are derived from three different experiments.

Techniques Used: Plasmid Preparation, Ligation, Agarose Gel Electrophoresis, Software, Derivative Assay

Monomeric form of TrfA protein uncuffs the RK2 handcuff structures. Schemes of the experimental procedure are presented in panel ( A ) NA assay, and ( C ) ligation assay. Uncuffing of the RK2 handcuff structures was tested on pre-assembled handcuff structures as described under Materials and methods. After handcuffing reaction the influence of monomeric TrfA form was analyzed in NA ( B ) and ligase ( D ) assays. On agarose gel free DNA and DNA multimers are marked F and M, respectively. A quantified densitometry with using ChemiDoc MP System and ImageLab™ Software (BioRad) were applied for DNA multimers amount estimation. The error bars are derived from three different experiments.
Figure Legend Snippet: Monomeric form of TrfA protein uncuffs the RK2 handcuff structures. Schemes of the experimental procedure are presented in panel ( A ) NA assay, and ( C ) ligation assay. Uncuffing of the RK2 handcuff structures was tested on pre-assembled handcuff structures as described under Materials and methods. After handcuffing reaction the influence of monomeric TrfA form was analyzed in NA ( B ) and ligase ( D ) assays. On agarose gel free DNA and DNA multimers are marked F and M, respectively. A quantified densitometry with using ChemiDoc MP System and ImageLab™ Software (BioRad) were applied for DNA multimers amount estimation. The error bars are derived from three different experiments.

Techniques Used: Ligation, Agarose Gel Electrophoresis, Software, Derivative Assay

Uncuffing of the RK2 handcuff structures is mediated by Lon and ClpAP proteases. The scheme of the experimental procedure is presented in panel ( A ) NA assay, and ( C ) ligation assay. Uncuffing of the RK2 handcuff structures was tested on pre-assembled handcuff structures as described in Materials and methods. After handcuffing reaction the influence of several chaperones (ClpX and ClpA) and proteases (ClpXP, ClpAP and Lon) was analyzed by the NA ( B ) and ligase ( D ) assays. On agarose gel free DNA and DNA multimers are marked F and M, respectively. Positive control (lane 1, B and D ) reactions with wild-type TrfA protein without the use of chaperones or proteases. Stability of TrfA protein, during analysis of handcuff structure disruption by chaperones and proteases in ligase assays, was tested by SDS-PAGE electrophoresis ( E ). A quantified densitometry with using ChemiDoc MP System and ImageLab™ Software (BioRad) were applied for TrfA protein and DNA multimers amount estimation. The error bars are derived from three different experiments.
Figure Legend Snippet: Uncuffing of the RK2 handcuff structures is mediated by Lon and ClpAP proteases. The scheme of the experimental procedure is presented in panel ( A ) NA assay, and ( C ) ligation assay. Uncuffing of the RK2 handcuff structures was tested on pre-assembled handcuff structures as described in Materials and methods. After handcuffing reaction the influence of several chaperones (ClpX and ClpA) and proteases (ClpXP, ClpAP and Lon) was analyzed by the NA ( B ) and ligase ( D ) assays. On agarose gel free DNA and DNA multimers are marked F and M, respectively. Positive control (lane 1, B and D ) reactions with wild-type TrfA protein without the use of chaperones or proteases. Stability of TrfA protein, during analysis of handcuff structure disruption by chaperones and proteases in ligase assays, was tested by SDS-PAGE electrophoresis ( E ). A quantified densitometry with using ChemiDoc MP System and ImageLab™ Software (BioRad) were applied for TrfA protein and DNA multimers amount estimation. The error bars are derived from three different experiments.

Techniques Used: Ligation, Agarose Gel Electrophoresis, Positive Control, SDS Page, Electrophoresis, Software, Derivative Assay

30) Product Images from "The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress"

Article Title: The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

Journal: eLife

doi: 10.7554/eLife.58020

RIF1 splice variants are expressed in vivo. RIF1 splice variants were amplified from a human cDNA panel covering 48 major tissues, using primers LW030 and LW031 that produce bands of 322 bp for RIF1-S and 400 bp for RIF1-L. The PCR products were analysed on a 2% agarose gel and the relative abundance of RIF1-L and RIF1-S were calculated based on the band intensities using ImageLab Software (Bio-Rad). ND – not detected.
Figure Legend Snippet: RIF1 splice variants are expressed in vivo. RIF1 splice variants were amplified from a human cDNA panel covering 48 major tissues, using primers LW030 and LW031 that produce bands of 322 bp for RIF1-S and 400 bp for RIF1-L. The PCR products were analysed on a 2% agarose gel and the relative abundance of RIF1-L and RIF1-S were calculated based on the band intensities using ImageLab Software (Bio-Rad). ND – not detected.

Techniques Used: In Vivo, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Software

31) Product Images from "Architecture of the chikungunya virus replication organelle"

Article Title: Architecture of the chikungunya virus replication organelle

Journal: bioRxiv

doi: 10.1101/2022.04.05.487153

Representative gels related to Fig. 3 . (A-C). Copelletation of nsP1 with multilamellar vesicles (MLVs) with varying percentages of the anionic phospholipids POPG (A), PI(4)P (B), and PI(4,5)P 2 (C) in a background of POPC and 20% cholesterol. The supernatant and pellet were run on 10% SDS-PAGE. After destaining the Coomassie stained gel, image was acquired with a Chemidoc Imaging System (Bio-Rad) and the relative intensity of bands were quantified using ImageLab software (Bio-Rad) and plotted as shown in Figure 3 (A-C) .
Figure Legend Snippet: Representative gels related to Fig. 3 . (A-C). Copelletation of nsP1 with multilamellar vesicles (MLVs) with varying percentages of the anionic phospholipids POPG (A), PI(4)P (B), and PI(4,5)P 2 (C) in a background of POPC and 20% cholesterol. The supernatant and pellet were run on 10% SDS-PAGE. After destaining the Coomassie stained gel, image was acquired with a Chemidoc Imaging System (Bio-Rad) and the relative intensity of bands were quantified using ImageLab software (Bio-Rad) and plotted as shown in Figure 3 (A-C) .

Techniques Used: SDS Page, Staining, Imaging, Software

32) Product Images from "SARS-CoV-2 Protein in Wastewater Mirrors COVID-19 Prevalence"

Article Title: SARS-CoV-2 Protein in Wastewater Mirrors COVID-19 Prevalence

Journal: medRxiv

doi: 10.1101/2020.09.01.20185280

SARS-CoV-2 membrane (M) protein is detectable in primary sludge from wastewater in Ottawa and Gatineau and tracks RNA. Protein was extracted from unconcentrated primary sludge samples from (A) Ottawa (May 5, 2020), (B) Ottawa (April 24, 2020) and (C) Gatineau (collected between April 16 and May 26, 2020), comparing RIPA, urea and triton X-100 lysis buffers. Protein lysates were subsequently cleaned up by acetone-precipitation (see methods and materials), resuspended in RIPA lysis buffer with loading dye and subsequently resolved by 10% SDS-PAGE for the detection of M protein . (D) Red circle: M protein signal at 25kDa (indicated by red arrow in Figure 3C ) for the Gatineau time-series was quantified by densitometric analysis using ImageLab software, normalized to total protein as detected by the stain-free signal, and plotted as the average level of protein detected by RIPA, urea, and triton X-100 extraction methods. Y-values are protein levels relative to levels detected on April 16 (set to a value of 1.0). Wastewater sample collection dates are specified on the X-axis. Error bars represent standard deviation between different extraction methods (RIPA, UREA and Triton X-100). Black triangles and squares (From D’Aoust et al ., 2020) 8 : quantification of viral RNA (N1 and N2, respectively) numbers from PEG precipitated primary sludge quantified by qPCR and normalized to PMMoV. Y-values are RNA levels relative to level on April 16 (set to a value of 1.0). Collection dates are specified on the X-axis.
Figure Legend Snippet: SARS-CoV-2 membrane (M) protein is detectable in primary sludge from wastewater in Ottawa and Gatineau and tracks RNA. Protein was extracted from unconcentrated primary sludge samples from (A) Ottawa (May 5, 2020), (B) Ottawa (April 24, 2020) and (C) Gatineau (collected between April 16 and May 26, 2020), comparing RIPA, urea and triton X-100 lysis buffers. Protein lysates were subsequently cleaned up by acetone-precipitation (see methods and materials), resuspended in RIPA lysis buffer with loading dye and subsequently resolved by 10% SDS-PAGE for the detection of M protein . (D) Red circle: M protein signal at 25kDa (indicated by red arrow in Figure 3C ) for the Gatineau time-series was quantified by densitometric analysis using ImageLab software, normalized to total protein as detected by the stain-free signal, and plotted as the average level of protein detected by RIPA, urea, and triton X-100 extraction methods. Y-values are protein levels relative to levels detected on April 16 (set to a value of 1.0). Wastewater sample collection dates are specified on the X-axis. Error bars represent standard deviation between different extraction methods (RIPA, UREA and Triton X-100). Black triangles and squares (From D’Aoust et al ., 2020) 8 : quantification of viral RNA (N1 and N2, respectively) numbers from PEG precipitated primary sludge quantified by qPCR and normalized to PMMoV. Y-values are RNA levels relative to level on April 16 (set to a value of 1.0). Collection dates are specified on the X-axis.

Techniques Used: Lysis, SDS Page, Software, Staining, Standard Deviation, Real-time Polymerase Chain Reaction

33) Product Images from "Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication"

Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

Journal: Heliyon

doi: 10.1016/j.heliyon.2018.e00525

A-E . The relative intensities of protein bands (mean ± SE) are shown in bar graphs for TLR4, connexin 43 (Cx 43), Na + /K + ATPase, GLT-1, GLAST-1. Intensity were quantified using the ImageLab software in the linear exposure range and normalized to β-actin from the same blot. The intensity of the control protein band was set to 1.
Figure Legend Snippet: A-E . The relative intensities of protein bands (mean ± SE) are shown in bar graphs for TLR4, connexin 43 (Cx 43), Na + /K + ATPase, GLT-1, GLAST-1. Intensity were quantified using the ImageLab software in the linear exposure range and normalized to β-actin from the same blot. The intensity of the control protein band was set to 1.

Techniques Used: Software

34) Product Images from "Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer"

Article Title: Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.14894

Effects of CDK8/19 inhibition on ER A . Luciferase expression from ER-dependent consensus promoter in T47D-ER/Luc reporter cells deprived of estrogen for 72 hours prior to treatment with 10 nM estradiol (E2) and increasing concentrations of Senexin A for 18 hours. RLU refers to relative luciferase units as a percentage of control. B . Western blot analysis of phospho-ER (Ser118) and ER expression in T47D-ER/Luc reporter cells deprived of estrogen for 72 hours prior to treatment with 10 nM estradiol (E2) in the presence or absence of 2.5 µM Senexin A for 15 mins and 6 hours. Loading was normalized using GAPDH and densitometry was performed using ImageLab software.
Figure Legend Snippet: Effects of CDK8/19 inhibition on ER A . Luciferase expression from ER-dependent consensus promoter in T47D-ER/Luc reporter cells deprived of estrogen for 72 hours prior to treatment with 10 nM estradiol (E2) and increasing concentrations of Senexin A for 18 hours. RLU refers to relative luciferase units as a percentage of control. B . Western blot analysis of phospho-ER (Ser118) and ER expression in T47D-ER/Luc reporter cells deprived of estrogen for 72 hours prior to treatment with 10 nM estradiol (E2) in the presence or absence of 2.5 µM Senexin A for 15 mins and 6 hours. Loading was normalized using GAPDH and densitometry was performed using ImageLab software.

Techniques Used: Inhibition, Luciferase, Expressing, Western Blot, Software

35) Product Images from "Cranberry Polyphenols in Esophageal Cancer Inhibition: New Insights"

Article Title: Cranberry Polyphenols in Esophageal Cancer Inhibition: New Insights

Journal: Nutrients

doi: 10.3390/nu14050969

Polyphenols modulate several proteins related to DNA damage, cell cycle and growth arrest in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL), AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to the appropriate loading control, GAPDH or HSP60, and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. FL and CL denote full-length and cleaved proteins, respectively. The plus (+) sign denotes treatment group.
Figure Legend Snippet: Polyphenols modulate several proteins related to DNA damage, cell cycle and growth arrest in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL), AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to the appropriate loading control, GAPDH or HSP60, and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. FL and CL denote full-length and cleaved proteins, respectively. The plus (+) sign denotes treatment group.

Techniques Used: Isolation, Western Blot, Expressing

Polyphenols modulate several proteins related to P53, inflammation and metastasis in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL, AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to the appropriate loading control, GAPDH or HSP60, and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. The asterisk (*) next to the P53 results in OE19 cells denote a faster migrating protein due to an insertion and duplication in the TP53 coding region as described in the results and discussion. FL and CL denote full-length and cleaved proteins, respectively. The plus (+) sign denotes treatment group.
Figure Legend Snippet: Polyphenols modulate several proteins related to P53, inflammation and metastasis in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL, AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to the appropriate loading control, GAPDH or HSP60, and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. The asterisk (*) next to the P53 results in OE19 cells denote a faster migrating protein due to an insertion and duplication in the TP53 coding region as described in the results and discussion. FL and CL denote full-length and cleaved proteins, respectively. The plus (+) sign denotes treatment group.

Techniques Used: Isolation, Western Blot, Expressing

Polyphenols modulate several proteins involved in inflammation, DNA damage and cell cycle in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL), AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to GAPDH and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. CL denotes a cleaved protein. The plus (+) sign denotes treatment group.
Figure Legend Snippet: Polyphenols modulate several proteins involved in inflammation, DNA damage and cell cycle in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL), AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to GAPDH and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. CL denotes a cleaved protein. The plus (+) sign denotes treatment group.

Techniques Used: Isolation, Western Blot, Expressing

36) Product Images from "Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington’s Disease and Human Blood Plasma"

Article Title: Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington’s Disease and Human Blood Plasma

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms23105598

Normalized intensity of total huntingtin in extracellular vesicles isolated from blood plasma of TgHD and KI-HD pig models and human plasma. EVs were isolated by ultracentrifugation and HTT forms were detected by western blot using anti-N-terminal HTT antibody recognizing both normal and mutant HTT. HTT band intensities were quantified by ImageLab software and normalized to CD9 levels to minimize variability in EV loading amount. Significantly higher total HTT content was found in vesicles from TgHD pigs compared to WT control pigs ( A ), while in KI-HD ( B ) and HD patients ( C ) the increase in total HTT in EVs compared to controls was not significant. The dots represent individual samples, bars show mean of the groups, and error bars indicate standard error of the mean. Statistical analysis using three-way ANOVA with genotype and age and sex as factors, followed by the post-hoc Tukey HSD test was performed for pig samples. T-test was used to compare HTT intensity between HD patient and control groups.
Figure Legend Snippet: Normalized intensity of total huntingtin in extracellular vesicles isolated from blood plasma of TgHD and KI-HD pig models and human plasma. EVs were isolated by ultracentrifugation and HTT forms were detected by western blot using anti-N-terminal HTT antibody recognizing both normal and mutant HTT. HTT band intensities were quantified by ImageLab software and normalized to CD9 levels to minimize variability in EV loading amount. Significantly higher total HTT content was found in vesicles from TgHD pigs compared to WT control pigs ( A ), while in KI-HD ( B ) and HD patients ( C ) the increase in total HTT in EVs compared to controls was not significant. The dots represent individual samples, bars show mean of the groups, and error bars indicate standard error of the mean. Statistical analysis using three-way ANOVA with genotype and age and sex as factors, followed by the post-hoc Tukey HSD test was performed for pig samples. T-test was used to compare HTT intensity between HD patient and control groups.

Techniques Used: Isolation, Western Blot, Mutagenesis, Software

37) Product Images from "Targeting CCN2 protects against progressive non-alcoholic steatohepatitis in a preclinical model induced by high-fat feeding and type 2 diabetes"

Article Title: Targeting CCN2 protects against progressive non-alcoholic steatohepatitis in a preclinical model induced by high-fat feeding and type 2 diabetes

Journal: Journal of Cell Communication and Signaling

doi: 10.1007/s12079-022-00667-1

Effects of CCN2Ab on CCN1 and CCN3 expression in high fat fed with diabetes mice. CCN1 ( A ), and CCN3 ( B ) mRNA quantitation. Detection of CCN1 and CCN3 by immunoblot; GAPDH was a protein loading control ( C ). Band intensity in the immunoblot was quantitated using ImageLab ( D and E ). Data is shown as Mean ± SEM as fold change of Chow + IgG. * p
Figure Legend Snippet: Effects of CCN2Ab on CCN1 and CCN3 expression in high fat fed with diabetes mice. CCN1 ( A ), and CCN3 ( B ) mRNA quantitation. Detection of CCN1 and CCN3 by immunoblot; GAPDH was a protein loading control ( C ). Band intensity in the immunoblot was quantitated using ImageLab ( D and E ). Data is shown as Mean ± SEM as fold change of Chow + IgG. * p

Techniques Used: Expressing, Mouse Assay, Quantitation Assay

Effects of CCN2Ab on MAPK signaling pathway in high fat fed with diabetes mice. A detection of phospho-ERK, total ERK by immunoblot; GAPDH is a protein loading control; C detection of phospho-p38, total p38 by immunoblot; ɑ-tubulin is a protein loading control; E . detection of phospho-JNK, total JNK by immunoblot; GAPDH is a protein loading control. Band intensity in the immunoblot was semi-quantitated using ImageLab ( B , D and F ). Data is mean ± SEM as fold change of Chow + IgG. * p
Figure Legend Snippet: Effects of CCN2Ab on MAPK signaling pathway in high fat fed with diabetes mice. A detection of phospho-ERK, total ERK by immunoblot; GAPDH is a protein loading control; C detection of phospho-p38, total p38 by immunoblot; ɑ-tubulin is a protein loading control; E . detection of phospho-JNK, total JNK by immunoblot; GAPDH is a protein loading control. Band intensity in the immunoblot was semi-quantitated using ImageLab ( B , D and F ). Data is mean ± SEM as fold change of Chow + IgG. * p

Techniques Used: Mouse Assay

38) Product Images from "RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit"

Article Title: RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1009583

RIOK2 is phosphorylated at Ser483 upon activation of the MAPK pathway. (A) HEK293 cells were transfected with a plasmid expressing HA-RIOK2 or an empty vector (Ctl). HA-RIOK2 was immunoprecipitated from serum-starved cells treated (+) or not (-) with PD184352 for 1h (10 μM) prior to PMA (100 ng/ml, 20 min) or EGF (25 μg/ml, 10 min) stimulation (+). Samples were analyzed by WB using anti-RXRXXpS/T or anti-HA antibodies. (B) RXRXXpS/T ((P)-RIOK2) and total RIOK2 signals obtained in (A) were quantified using ImageLab software and expressed as fold change relative to the PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (C) HA-RIOK2 was immunoprecipitated from serum-starved (Ctl) HEK293 cells treated (PMA+PD) or not (PMA) with PD184352 (10 μM, 1h) prior to PMA stimulation (100 ng/ml, 20 min). Purified HA-RIOK2 was isolated following SDS-PAGE, in gel digested with trypsin and the resulting peptides were submitted to nano-LC-MS/MS analysis. Label-free quantitative analysis of phosphorylation of the different RIOK2 phospho-peptides was performed as specified in the Materials and Methods section. Data are representative of triple biological replicate experiments for each condition. (D) HEK293 cells expressing HA-tagged versions of WT or mutant versions of RIOK2 (T481A or S483A) were serum starved (-) prior to PMA (100 ng/ml, 20 min) stimulation (+). HA-RIOK2 was immunoprecipitated and samples were analyzed by WB as in (A) . (E) RXRXXpS/T ((P)-RIOK2) and total RIOK2 signals obtained in (D) were quantified using ImageLab software and expressed as fold change relative to the WT + PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (F) Serum-starved HEK293 cells were stimulated with different agonists of the MAPK pathway. Phosphorylation of endogenous RIOK2 at Ser483 ((P)-RIOK2) was monitored by WB using specific antibodies. (G) (P)-RIOK2 and total RIOK2 signals obtained in (F) were quantified using ImageLab software and expressed as fold change relative to the starved condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test).
Figure Legend Snippet: RIOK2 is phosphorylated at Ser483 upon activation of the MAPK pathway. (A) HEK293 cells were transfected with a plasmid expressing HA-RIOK2 or an empty vector (Ctl). HA-RIOK2 was immunoprecipitated from serum-starved cells treated (+) or not (-) with PD184352 for 1h (10 μM) prior to PMA (100 ng/ml, 20 min) or EGF (25 μg/ml, 10 min) stimulation (+). Samples were analyzed by WB using anti-RXRXXpS/T or anti-HA antibodies. (B) RXRXXpS/T ((P)-RIOK2) and total RIOK2 signals obtained in (A) were quantified using ImageLab software and expressed as fold change relative to the PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (C) HA-RIOK2 was immunoprecipitated from serum-starved (Ctl) HEK293 cells treated (PMA+PD) or not (PMA) with PD184352 (10 μM, 1h) prior to PMA stimulation (100 ng/ml, 20 min). Purified HA-RIOK2 was isolated following SDS-PAGE, in gel digested with trypsin and the resulting peptides were submitted to nano-LC-MS/MS analysis. Label-free quantitative analysis of phosphorylation of the different RIOK2 phospho-peptides was performed as specified in the Materials and Methods section. Data are representative of triple biological replicate experiments for each condition. (D) HEK293 cells expressing HA-tagged versions of WT or mutant versions of RIOK2 (T481A or S483A) were serum starved (-) prior to PMA (100 ng/ml, 20 min) stimulation (+). HA-RIOK2 was immunoprecipitated and samples were analyzed by WB as in (A) . (E) RXRXXpS/T ((P)-RIOK2) and total RIOK2 signals obtained in (D) were quantified using ImageLab software and expressed as fold change relative to the WT + PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (F) Serum-starved HEK293 cells were stimulated with different agonists of the MAPK pathway. Phosphorylation of endogenous RIOK2 at Ser483 ((P)-RIOK2) was monitored by WB using specific antibodies. (G) (P)-RIOK2 and total RIOK2 signals obtained in (F) were quantified using ImageLab software and expressed as fold change relative to the starved condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test).

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Software, One-tailed Test, MANN-WHITNEY, Purification, Isolation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

RIOK2 is a direct RSK substrate. (A) Serum-starved (Ctl) HEK293 cells were treated or not (-) with PD184352 (PD), or LJH685 (10 μM) (LJH) for 1 h prior to PMA stimulation (100 ng/ml, 20 min). Phosphorylation of endogenous RIOK2 at Ser483 ((P)-RIOK2) was monitored by WB using specific antibodies. (B) (P)-RIOK2 and total RIOK2 signals obtained in (A) were quantified using ImageLab software and expressed as fold change relative to the PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (C) HEK293 cells were transfected with vectors over-expressing HA-tagged RSK1, RSK2, RSK3 or RSK4, or the empty vector (Ctl). Following serum-starvation and PMA stimulation (100 ng/ml, 20 min), samples were analyzed by WB using the indicated antibodies. (D) (P)-RIOK2 and total RIOK2 signals obtained in (C) were quantified using ImageLab software and expressed as fold change relative to starved Ctl cells. Statistically significant differences between starved conditions relative to starved Ctl cells are indicated by hashes, and between PMA stimulated conditions relative to stimulated Ctl cells by asterisks (#/*: P≤0.05, ns: not statistically significant, One-tailed Mann Whitney test). (E) HEK293 cells expressing shRNA targeting an irrelevant sequence (Ctl) or both RSK1 and RSK2 (RSK1/2) were processed as in (C) . (F) (P)-RIOK2 and total RIOK2 signals obtained in (E) were quantified using ImageLab software and expressed as fold change relative to the starved condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (G) Human activated RSK1 was incubated in the presence of γ[ 32 P]-ATP with either GST alone, or a GST-RIOK2 peptide (D443-E552) containing either S483 (RIOK2 WT ) or the non-phosphorylatable version (RIOK2 S483A ). The resulting samples were analyzed by SDS-PAGE and revealed by autoradiography or Coomassie blue staining. Quantification of [ 32 P] incorporation within each peptide is expressed as n-fold change compared to the absence of RSK1. (H) In vitro kinase assays performed as in (G) in the presence or not of RSK inhibitors BI-D1870 or LJH685 (10 mM).
Figure Legend Snippet: RIOK2 is a direct RSK substrate. (A) Serum-starved (Ctl) HEK293 cells were treated or not (-) with PD184352 (PD), or LJH685 (10 μM) (LJH) for 1 h prior to PMA stimulation (100 ng/ml, 20 min). Phosphorylation of endogenous RIOK2 at Ser483 ((P)-RIOK2) was monitored by WB using specific antibodies. (B) (P)-RIOK2 and total RIOK2 signals obtained in (A) were quantified using ImageLab software and expressed as fold change relative to the PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (C) HEK293 cells were transfected with vectors over-expressing HA-tagged RSK1, RSK2, RSK3 or RSK4, or the empty vector (Ctl). Following serum-starvation and PMA stimulation (100 ng/ml, 20 min), samples were analyzed by WB using the indicated antibodies. (D) (P)-RIOK2 and total RIOK2 signals obtained in (C) were quantified using ImageLab software and expressed as fold change relative to starved Ctl cells. Statistically significant differences between starved conditions relative to starved Ctl cells are indicated by hashes, and between PMA stimulated conditions relative to stimulated Ctl cells by asterisks (#/*: P≤0.05, ns: not statistically significant, One-tailed Mann Whitney test). (E) HEK293 cells expressing shRNA targeting an irrelevant sequence (Ctl) or both RSK1 and RSK2 (RSK1/2) were processed as in (C) . (F) (P)-RIOK2 and total RIOK2 signals obtained in (E) were quantified using ImageLab software and expressed as fold change relative to the starved condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (G) Human activated RSK1 was incubated in the presence of γ[ 32 P]-ATP with either GST alone, or a GST-RIOK2 peptide (D443-E552) containing either S483 (RIOK2 WT ) or the non-phosphorylatable version (RIOK2 S483A ). The resulting samples were analyzed by SDS-PAGE and revealed by autoradiography or Coomassie blue staining. Quantification of [ 32 P] incorporation within each peptide is expressed as n-fold change compared to the absence of RSK1. (H) In vitro kinase assays performed as in (G) in the presence or not of RSK inhibitors BI-D1870 or LJH685 (10 mM).

Techniques Used: Western Blot, Software, One-tailed Test, MANN-WHITNEY, Transfection, Expressing, Plasmid Preparation, shRNA, Sequencing, Incubation, SDS Page, Autoradiography, Staining, In Vitro

RIOK2 phosphorylation at Ser483 is required for efficient maturation of pre-40S particles. (A) MTS assays were performed on Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines at the indicated time points. ODs at 490 nm were measured using Spectramax. (B) Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines were incubated with 1 μM puromycin for the indicated times. Levels of puromycin-labelled peptides were monitored by WB using anti-puromycin antibodies. WB signals were quantified using ImageLab software and expressed as arbitrary units (a.u.). (C) , Total cellular RNAs were extracted from control (Ctl), RIOK2 S483A (1 to 3) or RIOK2 S483D eHAP1 cell lines. Accumulation levels of pre-rRNAs and mature rRNAs were analyzed by NB as in Fig 1 . (D) RAMP analyses of pre-rRNA levels obtained in (C) normalized to the 26S signals after quantification using MultiGauge software (Fujifilm). Graphical representations show fold changes compared to the control condition (Ctl). Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, Two-way ANOVA, Bonferroni posttests). (E) FISH experiments performed on RIOK2 WT (Ctl) and RIOK2 S483A eHAP1 cell lines. Pre-rRNAs were detected using a Cy5-labeled 5’-ITS1 probe. Cells were stained with DAPI to visualize nuclei, and images were captured in identical setting conditions. (F) Nucleolar, nuclear and cytoplasmic fluorescence signals were quantified using ImageJ software, as described in the “Materials and Methods” section and S7 Fig . Graph representations show fold change in RIOK2 S483A relative to RIOK2 WT cell line (n = 100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, *: P≤0.05, One-tailed Mann Whitney test).
Figure Legend Snippet: RIOK2 phosphorylation at Ser483 is required for efficient maturation of pre-40S particles. (A) MTS assays were performed on Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines at the indicated time points. ODs at 490 nm were measured using Spectramax. (B) Control (Ctl) and RIOK2 S483A (1 to 3) eHAP1 cell lines were incubated with 1 μM puromycin for the indicated times. Levels of puromycin-labelled peptides were monitored by WB using anti-puromycin antibodies. WB signals were quantified using ImageLab software and expressed as arbitrary units (a.u.). (C) , Total cellular RNAs were extracted from control (Ctl), RIOK2 S483A (1 to 3) or RIOK2 S483D eHAP1 cell lines. Accumulation levels of pre-rRNAs and mature rRNAs were analyzed by NB as in Fig 1 . (D) RAMP analyses of pre-rRNA levels obtained in (C) normalized to the 26S signals after quantification using MultiGauge software (Fujifilm). Graphical representations show fold changes compared to the control condition (Ctl). Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, Two-way ANOVA, Bonferroni posttests). (E) FISH experiments performed on RIOK2 WT (Ctl) and RIOK2 S483A eHAP1 cell lines. Pre-rRNAs were detected using a Cy5-labeled 5’-ITS1 probe. Cells were stained with DAPI to visualize nuclei, and images were captured in identical setting conditions. (F) Nucleolar, nuclear and cytoplasmic fluorescence signals were quantified using ImageJ software, as described in the “Materials and Methods” section and S7 Fig . Graph representations show fold change in RIOK2 S483A relative to RIOK2 WT cell line (n = 100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, *: P≤0.05, One-tailed Mann Whitney test).

Techniques Used: Incubation, Western Blot, Software, Fluorescence In Situ Hybridization, Labeling, Staining, Fluorescence, One-tailed Test, MANN-WHITNEY

RIOK2 phosphorylation at Ser483 facilitates its release from pre-40S particles and re-import into the nucleus. (A) HEK293 cells were co-transfected with plasmids expressing HA-NOB1 and Flag-RIOK2 WT , Flag-RIOK2 S483A or Flag-RIOK2 S483D . Pre-40S particles were immunopurified via HA-NOB1 and subsequently incubated for 45 or 90 min with a buffer inducing RIOK2 release at 16°C. The presence of RIOK2, LTV1 and RPS7 proteins in supernatants (released proteins) and on beads (pre-40S-bound proteins) were analyzed by WB. Experiments with Flag-RIOK2 S483A and Flag-RIOK2 S483D were performed with different sets of Flag-RIOK2 WT as controls. A representative WB experiment for Flag-RIOK2 WT is shown. (B) Quantification of WB signals from (A) using ImageLab software and expressed as released over bound RIOK2 ratios. Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, Two-way ANOVA test, Bonferroni posttests). (C) RIOK2 WT and RIOK2 S483A eHAP1 cells were treated with Leptomycin B (LMB, 20 nM) for the indicated times. Subcellular localization of RIOK2 was monitored by immunofluorescence microscopy using specific antibodies. Nuclei were visualized by DAPI staining. (D) , Quantification of nuclear to cytoplasmic fluorescence ratios at the indicated time points obtained in (C) using ImageJ software (n = 100 cells from different fields), as described in S7 Fig . Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, 2way ANOVA tests, Bonferroni posttests).
Figure Legend Snippet: RIOK2 phosphorylation at Ser483 facilitates its release from pre-40S particles and re-import into the nucleus. (A) HEK293 cells were co-transfected with plasmids expressing HA-NOB1 and Flag-RIOK2 WT , Flag-RIOK2 S483A or Flag-RIOK2 S483D . Pre-40S particles were immunopurified via HA-NOB1 and subsequently incubated for 45 or 90 min with a buffer inducing RIOK2 release at 16°C. The presence of RIOK2, LTV1 and RPS7 proteins in supernatants (released proteins) and on beads (pre-40S-bound proteins) were analyzed by WB. Experiments with Flag-RIOK2 S483A and Flag-RIOK2 S483D were performed with different sets of Flag-RIOK2 WT as controls. A representative WB experiment for Flag-RIOK2 WT is shown. (B) Quantification of WB signals from (A) using ImageLab software and expressed as released over bound RIOK2 ratios. Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, Two-way ANOVA test, Bonferroni posttests). (C) RIOK2 WT and RIOK2 S483A eHAP1 cells were treated with Leptomycin B (LMB, 20 nM) for the indicated times. Subcellular localization of RIOK2 was monitored by immunofluorescence microscopy using specific antibodies. Nuclei were visualized by DAPI staining. (D) , Quantification of nuclear to cytoplasmic fluorescence ratios at the indicated time points obtained in (C) using ImageJ software (n = 100 cells from different fields), as described in S7 Fig . Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, 2way ANOVA tests, Bonferroni posttests).

Techniques Used: Transfection, Expressing, Incubation, Western Blot, Software, Immunofluorescence, Microscopy, Staining, Fluorescence

39) Product Images from "Nomad Jellyfish Rhopilema nomadica Venom Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Hepatocellular Carcinoma HepG2 Cells"

Article Title: Nomad Jellyfish Rhopilema nomadica Venom Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Hepatocellular Carcinoma HepG2 Cells

Journal: Molecules

doi: 10.3390/molecules26175185

Western blot analysis. ( A ) shows western blot results about the expression level of apoptosis-related proteins BAX, Bcl-2, caspase-3, and p53 in HepG2 cells after being treated for 48 h with IC 50 concentration of R. nomadica venom. From ( B – F ) represent the statistical graphs of the density ratios of the proteins calculated by ImageLab. Protein levels were normalized to β-Actin. Data are analyzed with paired Student’s t-test Values are represented as mean ± SEM. * denotes significance difference ( p
Figure Legend Snippet: Western blot analysis. ( A ) shows western blot results about the expression level of apoptosis-related proteins BAX, Bcl-2, caspase-3, and p53 in HepG2 cells after being treated for 48 h with IC 50 concentration of R. nomadica venom. From ( B – F ) represent the statistical graphs of the density ratios of the proteins calculated by ImageLab. Protein levels were normalized to β-Actin. Data are analyzed with paired Student’s t-test Values are represented as mean ± SEM. * denotes significance difference ( p

Techniques Used: Western Blot, Expressing, Concentration Assay

40) Product Images from "CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis"

Article Title: CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

Journal: Scientific Reports

doi: 10.1038/s41598-017-18442-3

Transient transfection, selection and screening for nanoluciferase repair. ( A ) Cartoon representation of transient nanoluciferase repair activity assay followed by selection with puromycin and G418. ( B ) A total of 5 × 10 7 parasites were nucleofected with 10 μg of pCas9-gRNA( nluc ), 10 μg pMN:: nluc(stop) and 100 μg of either 50 bp or 125 bp annealed repair oligos or water using T-cell buffer (Lonza) and the U-033 program (Amaxa). Parasites were immediately recovered in completed TYM media + 1 μM Shield-1. After 24 hours of recovery, equivalent numbers of parasites (1 × 10 6 ) were assayed for nanoluciferase activity and the graph represents the average relative luminescence value +/− standard deviation from duplicate samples for each condition ( C ). After 24 hours of recovery, one transfection population of the pMPAC::EV- and pCas9-gRNA( nluc )-treated parasites from Fig. 4B was treated with 30 μg/ml puromycin and 50 μg/ml G418 to select for parasites transfected with both plasmids. After 7 and 14 days (post-nucleofection), parasites were re-tested for nanoluciferase activity by assay of equivalent numbers of parasites (1 × 10 6 ). The graph represents the average relative luminescence value +/− standard deviation for each condition. Inset: Anti-HA epitope immunoblot analysis of control samples (pre-selected parasites) compared to protein isolated from puromycin/G418 selected parasites grown for two weeks. ( D ) Upper: anti-HA epitope immunoblot analysis of controls (pre-selected plasmids) and representative clones from 14 day puromycin/G418 selected pCas9-gRNA( nluc ) + pMN:: nluc(stop) + 125 bp oligo parasites in Fig. 4C. Lower: representative DNA sequencing trace (Genewiz sequencing and FinchTV sequence viewer) of PCR products amplifying the nanoluciferase gene from T. vaginalis genomic DNA preparations. Highlighted (and arrows) are residues G50C and A54T which are the site of the two predicted modifications for repair of the stop codon (A54T) and oligo-modification confirmatory mutation (G50C). Immunoblot analyses in Fig. 4C and D were imaged using a Bio-Rad Gel Doc and ImageLab software. Full length blots/gels are presented in Supplementary Figure S5 .
Figure Legend Snippet: Transient transfection, selection and screening for nanoluciferase repair. ( A ) Cartoon representation of transient nanoluciferase repair activity assay followed by selection with puromycin and G418. ( B ) A total of 5 × 10 7 parasites were nucleofected with 10 μg of pCas9-gRNA( nluc ), 10 μg pMN:: nluc(stop) and 100 μg of either 50 bp or 125 bp annealed repair oligos or water using T-cell buffer (Lonza) and the U-033 program (Amaxa). Parasites were immediately recovered in completed TYM media + 1 μM Shield-1. After 24 hours of recovery, equivalent numbers of parasites (1 × 10 6 ) were assayed for nanoluciferase activity and the graph represents the average relative luminescence value +/− standard deviation from duplicate samples for each condition ( C ). After 24 hours of recovery, one transfection population of the pMPAC::EV- and pCas9-gRNA( nluc )-treated parasites from Fig. 4B was treated with 30 μg/ml puromycin and 50 μg/ml G418 to select for parasites transfected with both plasmids. After 7 and 14 days (post-nucleofection), parasites were re-tested for nanoluciferase activity by assay of equivalent numbers of parasites (1 × 10 6 ). The graph represents the average relative luminescence value +/− standard deviation for each condition. Inset: Anti-HA epitope immunoblot analysis of control samples (pre-selected parasites) compared to protein isolated from puromycin/G418 selected parasites grown for two weeks. ( D ) Upper: anti-HA epitope immunoblot analysis of controls (pre-selected plasmids) and representative clones from 14 day puromycin/G418 selected pCas9-gRNA( nluc ) + pMN:: nluc(stop) + 125 bp oligo parasites in Fig. 4C. Lower: representative DNA sequencing trace (Genewiz sequencing and FinchTV sequence viewer) of PCR products amplifying the nanoluciferase gene from T. vaginalis genomic DNA preparations. Highlighted (and arrows) are residues G50C and A54T which are the site of the two predicted modifications for repair of the stop codon (A54T) and oligo-modification confirmatory mutation (G50C). Immunoblot analyses in Fig. 4C and D were imaged using a Bio-Rad Gel Doc and ImageLab software. Full length blots/gels are presented in Supplementary Figure S5 .

Techniques Used: Transfection, Selection, Activity Assay, Standard Deviation, Isolation, Clone Assay, DNA Sequencing, Sequencing, Polymerase Chain Reaction, Modification, Mutagenesis, Software

Setup of pMN:: fkbp-cas9 and pMN::gRNA constructs. ( A ) Cartoon representation (not to scale) of pMN:: fkbp-cas9 plasmid. The T. vaginalis gene α-SCS 5′ and 3′ UTRs of the pMasterNEO plasmid (pMN) 53 drive expression of the fkbp (FKBP destabilization domain)- cas9 fusion gene. The cas9 variant used is the human codon optimized version with 2 SV40 nuclear localization signals at the C-terminus 56 . ( B ) Representative immunoblot analysis (inset) and quantification of replicate samples of pMN:: fkbp-cas9 expression. Samples were induced for 24 hours with 0, 0.1, 0.5 or 1 μM Shield-1 ligand before protein was collected and resolved by SDS-PAGE. Inset, upper: anti-Cas9 immunoblot (Clontech); Inset, lower: anti-TvGAPDH antibody (Cocalico Biologicals). Control sample used a pMN::EV (pMasterNEO::empty vector) transfectant. Quantitative comparison of samples utilized normalized signal of Cas9 (upper blot) to GAPDH signal (lower blot) taken from within an experiment and analyzed in parallel on separate immunoblots using a Bio-Rad Gel Doc and ImageLab software. Bar graph represents average +/− standard deviation of two independent analyses. ( C ) Cartoon representation (not to scale) of pMN:: gRNA – 360 bp of the T. vaginalis U6 5′ UTR and 37 bp of the 3′ UTR flank the gRNA. A 20 nt seed region with the first nucleotide always an adenine residue followed by 19 nt seed region used for specific targeting and the gRNA scaffold. ( D ) RT-PCR products amplifying either the gRNA sequence or the U6 snRNA (control). Total RNA was subjected to +/− reverse transcriptase then amplified by PCR using gRNA- or U6 snRNA-specific primers. Control PCRs included the pMN::EV and pMN:: gRNA plasmids, genomic DNA and no DNA template. PCR products were imaged using a Bio-Rad Gel Doc and ImageLab software. Full length blots/gels are presented in Supplementary Figure S5 .
Figure Legend Snippet: Setup of pMN:: fkbp-cas9 and pMN::gRNA constructs. ( A ) Cartoon representation (not to scale) of pMN:: fkbp-cas9 plasmid. The T. vaginalis gene α-SCS 5′ and 3′ UTRs of the pMasterNEO plasmid (pMN) 53 drive expression of the fkbp (FKBP destabilization domain)- cas9 fusion gene. The cas9 variant used is the human codon optimized version with 2 SV40 nuclear localization signals at the C-terminus 56 . ( B ) Representative immunoblot analysis (inset) and quantification of replicate samples of pMN:: fkbp-cas9 expression. Samples were induced for 24 hours with 0, 0.1, 0.5 or 1 μM Shield-1 ligand before protein was collected and resolved by SDS-PAGE. Inset, upper: anti-Cas9 immunoblot (Clontech); Inset, lower: anti-TvGAPDH antibody (Cocalico Biologicals). Control sample used a pMN::EV (pMasterNEO::empty vector) transfectant. Quantitative comparison of samples utilized normalized signal of Cas9 (upper blot) to GAPDH signal (lower blot) taken from within an experiment and analyzed in parallel on separate immunoblots using a Bio-Rad Gel Doc and ImageLab software. Bar graph represents average +/− standard deviation of two independent analyses. ( C ) Cartoon representation (not to scale) of pMN:: gRNA – 360 bp of the T. vaginalis U6 5′ UTR and 37 bp of the 3′ UTR flank the gRNA. A 20 nt seed region with the first nucleotide always an adenine residue followed by 19 nt seed region used for specific targeting and the gRNA scaffold. ( D ) RT-PCR products amplifying either the gRNA sequence or the U6 snRNA (control). Total RNA was subjected to +/− reverse transcriptase then amplified by PCR using gRNA- or U6 snRNA-specific primers. Control PCRs included the pMN::EV and pMN:: gRNA plasmids, genomic DNA and no DNA template. PCR products were imaged using a Bio-Rad Gel Doc and ImageLab software. Full length blots/gels are presented in Supplementary Figure S5 .

Techniques Used: Construct, Plasmid Preparation, Expressing, Variant Assay, SDS Page, Transfection, Western Blot, Software, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Polymerase Chain Reaction

CRISPR-directed KO of ferredoxin-1 and mif . ( A ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance gene flanked by the ferredoxin-1 UTRs and a plasmid containing Cas9 and Fd gRNA (pCas9-gRNA( Fd )) followed by selection for G418-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( B ) PCR analysis of G418-resistant clones for the presence of the neo resistance gene in the ferredoxin-1 locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5 A) combined with primers specific to the ferredoxin-1 locus (blue arrows in 5 A) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1665 bp and the 3′ PCR screen (middle panel) predicts a product of 1236 bp if the neo gene is present in the ferredoxin-1 locus. Lowest panel: PCR analysis of the ferredoxin-1 gene in clones. Amplification utilized primers specific to the wild-type ferredoxin-1 (yielding a 324 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( C ) Immunoblot protein analysis of the clones yielding positive PCR products for KO in Fig. 5B. Total protein extracts were resolved by SDS-PAGE and immunoblotted using anti-Ferredoxin-1 and TvGAPDH antibodies. All immunoblots contained equal amounts of the same experimental samples and were analyzed in parallel using a Bio-Rad Gel Doc and ImageLab software. ( D ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance (NeoR) gene flanked by the mif UTRs and a plasmid containing Cas9 and mif gRNAs (pCas9-gRNA( mif )), followed by selection for G418- and puromycin-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( E ) PCR analysis of G418 and puromycin-resistant clones for the presence of the neo resistance gene in the mif locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5D) combined with primers specific to the mif locus (orange arrows in 5D) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1290 bp and the 3′ PCR screen (middle panel) predicts a product of 1220 bp if the neo gene is present in the mif locus. Lowest panel: PCR analysis of the mif gene in clones. Amplification utilized primers specific to the wild-type mif gene (yielding a 500 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( F ) Immunoblot protein analysis of the clones yielding positive PCR products for KO in Fig. 5E. Total protein extracts were resolved by SDS-PAGE and immunoblotted using anti-MIF and TvGAPDH antibodies. All immunoblots contained equal amounts of the same experimental samples and were analyzed in parallel using a Bio-Rad Gel Doc and ImageLab software. Full length blots/gels are presented in Supplementary Figure S5 .
Figure Legend Snippet: CRISPR-directed KO of ferredoxin-1 and mif . ( A ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance gene flanked by the ferredoxin-1 UTRs and a plasmid containing Cas9 and Fd gRNA (pCas9-gRNA( Fd )) followed by selection for G418-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( B ) PCR analysis of G418-resistant clones for the presence of the neo resistance gene in the ferredoxin-1 locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5 A) combined with primers specific to the ferredoxin-1 locus (blue arrows in 5 A) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1665 bp and the 3′ PCR screen (middle panel) predicts a product of 1236 bp if the neo gene is present in the ferredoxin-1 locus. Lowest panel: PCR analysis of the ferredoxin-1 gene in clones. Amplification utilized primers specific to the wild-type ferredoxin-1 (yielding a 324 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( C ) Immunoblot protein analysis of the clones yielding positive PCR products for KO in Fig. 5B. Total protein extracts were resolved by SDS-PAGE and immunoblotted using anti-Ferredoxin-1 and TvGAPDH antibodies. All immunoblots contained equal amounts of the same experimental samples and were analyzed in parallel using a Bio-Rad Gel Doc and ImageLab software. ( D ) Cartoon representation of the KO scheme using nucleofection of parasites with a linearized neo resistance (NeoR) gene flanked by the mif UTRs and a plasmid containing Cas9 and mif gRNAs (pCas9-gRNA( mif )), followed by selection for G418- and puromycin-resistance (image not to scale). The resulting parasites were then screened for the presence of the KO and wt alleles using sequence-specific primers (horizontal arrows). ( E ) PCR analysis of G418 and puromycin-resistant clones for the presence of the neo resistance gene in the mif locus. PCR screens utilized primers specific to the neo resistance gene (yellow arrows in 5D) combined with primers specific to the mif locus (orange arrows in 5D) in regions outside the region present in the original KO PCR introduced by nucleofection. The 5′ end PCR screen (upper panel) predicts a product of 1290 bp and the 3′ PCR screen (middle panel) predicts a product of 1220 bp if the neo gene is present in the mif locus. Lowest panel: PCR analysis of the mif gene in clones. Amplification utilized primers specific to the wild-type mif gene (yielding a 500 bp product, if present). The PCR products for the different panels were run in parallel from reactions on 100 ng of the indicated genomic DNA extracts and resolved on separate agarose gels then imaged using a Bio-Rad Gel Doc and ImageLab software. ( F ) Immunoblot protein analysis of the clones yielding positive PCR products for KO in Fig. 5E. Total protein extracts were resolved by SDS-PAGE and immunoblotted using anti-MIF and TvGAPDH antibodies. All immunoblots contained equal amounts of the same experimental samples and were analyzed in parallel using a Bio-Rad Gel Doc and ImageLab software. Full length blots/gels are presented in Supplementary Figure S5 .

Techniques Used: CRISPR, Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Clone Assay, Amplification, Software, SDS Page, Western Blot

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Bio-Rad imagelab software
    Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. <t>ImageLab</t> software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P
    Imagelab Software, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/imagelab software/product/Bio-Rad
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    imagelab software - by Bioz Stars, 2022-09
    99/100 stars
      Buy from Supplier

    99
    Bio-Rad chemidoc imager
    Regulation of Glo1 activity via tyrosine 136 phosphorylation. (A) Mutation of Glo1 Y136 to alanine reduces Glo1 activity. Glo1 knockout HeLa cells were used to express 9 different point mutants of Glo1 or the wildtype protein as a control. Glo1 protein levels were quantified by immunoblotting and imaging with a <t>Chemidoc</t> (lower panel) and used to normalize Glo1 activity measurements, to arrive at Glo1 specific activity (upper panel). n = 4, ∗p
    Chemidoc Imager, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chemidoc imager/product/Bio-Rad
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chemidoc imager - by Bioz Stars, 2022-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

    doi: 10.3892/etm.2018.6646

    Figure Lengend Snippet: Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P

    Article Snippet: Relative quantification of proteins was performed using ImageLab software (version 4.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

    Techniques: Western Blot, Expressing, Transfection, Software

    p53's N-terminus as binding partner for POLι. K562 cells were transfected with expression plasmids for p53(WT), p53(22Q/23S) or empty vector (ctrl). Immunoprecipitations were performed 48 h after transfection. Pull-downs engaged polyclonal rabbit POLι-antibody (Bethyl, A301–304A) or control rabbit IgG, subsequent immunoblotting relied on anti-POLι, anti-p53 (mix of mAbs Pab421 and DO1) and light chain-specific peroxidase-coupled secondary antibody. The left panel shows a representative Western Blot. The right panel shows the quantification of three immunoprecipitations. Quantification of p53 was carried out with ImageLab software and corrected for values of the pull-down protein POLι, whereby the mean value for p53(WT) samples was set to 1. Mean ±SD. Note, that the values of the ctrl were subtracted from the values of p53(WT) and p53(22Q/23S). Due to the small number of values (n = 3) no statistical significant differences were achieved when comparing p53(WT) to p53(22Q/23S) or ctrl. IP = Immunoprecipitation of POLι.

    Journal: Nucleic Acids Research

    Article Title: Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance

    doi: 10.1093/nar/gkaa974

    Figure Lengend Snippet: p53's N-terminus as binding partner for POLι. K562 cells were transfected with expression plasmids for p53(WT), p53(22Q/23S) or empty vector (ctrl). Immunoprecipitations were performed 48 h after transfection. Pull-downs engaged polyclonal rabbit POLι-antibody (Bethyl, A301–304A) or control rabbit IgG, subsequent immunoblotting relied on anti-POLι, anti-p53 (mix of mAbs Pab421 and DO1) and light chain-specific peroxidase-coupled secondary antibody. The left panel shows a representative Western Blot. The right panel shows the quantification of three immunoprecipitations. Quantification of p53 was carried out with ImageLab software and corrected for values of the pull-down protein POLι, whereby the mean value for p53(WT) samples was set to 1. Mean ±SD. Note, that the values of the ctrl were subtracted from the values of p53(WT) and p53(22Q/23S). Due to the small number of values (n = 3) no statistical significant differences were achieved when comparing p53(WT) to p53(22Q/23S) or ctrl. IP = Immunoprecipitation of POLι.

    Article Snippet: Chemiluminescence detection and quantification of protein levels were carried out in the linear range using ImageLab software on a ChemiDocMP System (BioRad).

    Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Software, Immunoprecipitation

    Effect of leaves EtAc and ButOH extracts on LDLR and PCSK9 expression in Huh7 cell line. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p

    Journal: Food Chemistry: Molecular Sciences

    Article Title: Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food

    doi: 10.1016/j.fochms.2022.100126

    Figure Lengend Snippet: Effect of leaves EtAc and ButOH extracts on LDLR and PCSK9 expression in Huh7 cell line. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p

    Article Snippet: The densitometric readings were evaluated using ImageLab™ software (Bio-Rad, Segrate, Milan, Italy).

    Techniques: Expressing, Western Blot, Software, Quantitative RT-PCR

    Effect of fruit extract on LDLR and PCSK9 expression in Huh7 cells. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C) and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p

    Journal: Food Chemistry: Molecular Sciences

    Article Title: Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food

    doi: 10.1016/j.fochms.2022.100126

    Figure Lengend Snippet: Effect of fruit extract on LDLR and PCSK9 expression in Huh7 cells. A) and B) LDLR and PCSK9 expression was evaluated by western blot analysis. GAPDH was used as a loading control. Densitometric readings were evaluated using the ImageLab™ software, and the relative intensity of the bands are shown in the histograms. C) and D) LDLR and PCSK9 mRNA expression was evaluated by RT-qPCR analysis and normalized with 18S. *p

    Article Snippet: The densitometric readings were evaluated using ImageLab™ software (Bio-Rad, Segrate, Milan, Italy).

    Techniques: Expressing, Western Blot, Software, Quantitative RT-PCR

    Regulation of Glo1 activity via tyrosine 136 phosphorylation. (A) Mutation of Glo1 Y136 to alanine reduces Glo1 activity. Glo1 knockout HeLa cells were used to express 9 different point mutants of Glo1 or the wildtype protein as a control. Glo1 protein levels were quantified by immunoblotting and imaging with a Chemidoc (lower panel) and used to normalize Glo1 activity measurements, to arrive at Glo1 specific activity (upper panel). n = 4, ∗p

    Journal: Molecular Metabolism

    Article Title: The activity of glyoxylase 1 is regulated by glucose-responsive phosphorylation on Tyr136

    doi: 10.1016/j.molmet.2021.101406

    Figure Lengend Snippet: Regulation of Glo1 activity via tyrosine 136 phosphorylation. (A) Mutation of Glo1 Y136 to alanine reduces Glo1 activity. Glo1 knockout HeLa cells were used to express 9 different point mutants of Glo1 or the wildtype protein as a control. Glo1 protein levels were quantified by immunoblotting and imaging with a Chemidoc (lower panel) and used to normalize Glo1 activity measurements, to arrive at Glo1 specific activity (upper panel). n = 4, ∗p

    Article Snippet: A ChemiDoc imager (Biorad) was used for detecting and quantifying the signal.

    Techniques: Activity Assay, Mutagenesis, Knock-Out, Imaging