Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Subcellular colocalization of 24K and miRNA biogenesis proteins. ( A ) (i) Colocalization of 24K:mRFP with CFP:AtDCL1 in nuclear aggregates. ( B ) (i) Colocalization of 24K:mRFP with AtHYL1:YFP in nuclear aggregates. ( C ) (i) Colocalization of 24K:mRFP with AtSE:YFP in nuclear aggregates. (ii) The mRFP control does not colocalize in nuclear aggregates with any of the tested proteins. ( D ) Western blot analyses of extracts from N. benthamiana leaves in colocalization assays at 3 dpa. Anti-RFP (@RFP) or anti-GFP (@GFP) monoclonal antibodies were used for 24K or biogenesis proteins, respectively. Coomassie blue stain is shown as loading control. The numbers below correspond to normalized band density. Ratios between AtDCL and AtDCL1/24K (240 kDa); AtHYL1 and AtHYL1/24K (75 kDa); AtSE and AtSE/24K (110 kDa) are shown under the lines. Scale bar: 10 µm.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: Control, Western Blot, Bioprocessing, Staining

Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Interaction between 24K protein and miRNA biogenesis proteins. ( A ) In vivo analysis of BiFC assays in epidermal cells of N. benthamiana plants. The merge panels show positive interaction between N-mCitrine:24K and C-mCitrine:AtHYL1 (i) and N-mCitrine:24K and C-mCitrine:AtSE (ii) and no interaction between N-mCitrine:24K with C-mCitrine:AtDCL1 (iii). (iv). Negative control: N-mCitrine:24K + C-mCitrine (empty vector). Chloroplasts were marked in blue in the case of the negative interactions. In all cases, Fib:mRFP was used as an expression control of infiltrated samples. Scale bar: 10 µm. ( B ) Co-IP assay between 24K:mRFP and AtHYL1. INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins. (−) corresponds to the negative control using beads without @HYL1 antibody. (+) corresponds to the immunoprecipitated samples with @HYL1 attached to the beads. The anti-RFP (@RFP) and anti-HYL1 (@HYL1) monoclonal antibodies were used in each case to develop the blots. The presence of both proteins in the IP fraction indicates a positive interaction. ( C ) Co-IP assay between the 24K:mRFP and AtSE:YFP protein. The INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins with @GFP attached to the beads. Free mRFP was used as a negative control. The anti-RFP (@RFP) and anti-GFP (@GFP) antibodies were used in each case to develop the blots. The presence of both proteins in IP fraction indicates a positive interaction.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: In Vivo, Negative Control, Plasmid Preparation, Expressing, Control, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Bioprocessing

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: I427E mutation of αH in surface 2 of ILK-pKD impairs binding of GFP–ILK to GST–kindlin-2 F2PH. (A) GFP or GFP–ILK co-expressed with FLAG–α-parvin in CHO cells bound to glutathione bead-immobilized GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) as a negative control detected by immunoblotting. One representative blot for each construct tested is shown. The lane labeled ‘3%’ indicates 3% of input lysate. Bead loading was visualized by Ponceau S staining. (B) Quantification of GFP or GFP–ILK binding to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n=4). (C) Representative immunoblots for pulldown of GFP–ILK mutants co-expressed with FLAG–α-parvin in CHO cell lysates by GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A. The lane labeled ‘3%’ indicates 3% of input lysate. Bead loading was visualized by Ponceau S staining. (D) Quantification of binding of GFP–ILK and GFP–ILK mutants to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n≥3); *P<0.005 (Student's t-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots).

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Mutagenesis, Binding Assay, Negative Control, Western Blot, Construct, Labeling, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: Additional residues on αH in the ILK-pKD are implicated in the interaction with kindlin-2. (A) Ribbon diagram of helix-αH and surrounding residues in the ILK-pKD–α-parvin-CH2 co-crystal structure (PDB ID: 3KMW) generated with Chimera software (Pettersen et al., 2004). Residues selected for mutation are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK mutants by GST–kindlin-2 F2PH and GST–kindlin-2 F2PH L357A (L/A) from CHO cell lysate co-overexpressing FLAG–α-parvin assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n≥3; *P<0.001 (Student's t-test). (D,E) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 329-368 or GST–kindlin-2 329-368 L/A were assessed by representative immunoblots (D) and quantified (E); mean±s.e.m.; n≥3; *P≤0.0006. (F,G) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressing FLAG–α-parvin using GST or GST–kindlin-2 were assessed in representative immunoblots (F) and quantified (G); mean±s.e.m.; n=4; *P≤0.0001 (Student's t-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots). GST- protein loading is indicated by Ponceau S staining.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Generated, Software, Mutagenesis, Labeling, Western Blot, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: R243G/R334G double mutation of GFP–ILK (GFP–ILK RR/GG) impairs binding of the ILK to α-parvin. (A) Ribbon diagram of selected regions in the ILK KD–α-parvin-CH2 complex co-crystal structure (PDB ID: 3KMW) surrounding I244, F245, and S246, generated with Chimera software (Pettersen et al., 2004). Residues selected for mutagenesis are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK RR/GG from CHO cell lysates co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n=3; *P≤0.0001 (Student's t-test). (D) Diagram of the GFP nanotrap experiment. GFP–ILK was purified from lysate of cells co-expressing GFP–ILK and FLAG-α-parvin. The amount of co-purifying FLAG–α-parvin was assessed by immunoblotting and the FLAG:GFP ratio for each construct was calculated. (E,F) GFP-nanotrap co-purification of GFP–ILK constructs co-expressed FLAG–α-parvin in CHO cells was assessed by immunoblot from one experiment (E) and quantified (F) as a raw FLAG:GFP ratio. Dots represent single data points for each construct tested. The lane labeled ‘2%’ indicates the 2% input of lysate. (G) The FLAG:GFP ratio for each GFP or GFP–ILK construct tested is expressed relative to the FLAG:GFP ratio of the GFP–ILK control within each experiment, which is set to 1. The dataset includes the experiment shown in E and F (mean±s.e.m.; n≥4); *P≤0.0015; ns, statistically not significant, P>0.05 (Student's t-test). (H,I) Pulldown of GFP–ILK K220M from CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A assessed by representative immunoblot (H) and quantified (I); mean±s.e.m.; n=3; *P≤0.0001. Pulldown and co-purification quantification graphs are shown as bar charts with individual data points plotted (dots). GST-protein loading control for pulldown experiments is indicated by Ponceau S staining.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Mutagenesis, Binding Assay, Generated, Software, Labeling, Western Blot, Purification, Expressing, Construct, Copurification, Control, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: GFP–ILK mutants that are impaired in kindlin-2 binding localize poorly to focal adhesions. CHO cells stably expressing mCherry-paxillin were transiently co-transfected with FLAG–α-parvin and either GFP alone, GFP–ILK or one of the GFP–ILK mutants. Six hours after replating on fibronectin-coated glass-bottom dishes, live cells were imaged by epifluorescence (EPI) and/or TIRF microscopy as indicated. Images in each channel were linearly and uniformly adjusted, and cropped for clarity. Scale bar: 20 µm.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Microscopy

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: The ILK–kindlin-2 interaction is important for normal cell spreading in HeLa cells. (A) Immunoblotting of shScr and shILK HeLa cells to show protein levels of ILK, kindlin-2, α-parvin, PINCH1 and vinculin. (B) Bar graph showing residual protein levels in shILK cells calculated relative to those in shScr cells (mean±s.e.m.); individual data points are indicated (dots; n≥4). (C) Immunofluorescence staining of endogenous vinculin in fixed shScr or shILK HeLa cells spread on fibronectin-coated glass coverslips and acquired by epifluorescence microscopy. Scale bars: 20 µm. (D) CellProfiler quantification of GFP-positive cell areas pooled across three independent experiments shown as box and whiskers plots indicating 10th and 90th percentile range. n=155 shScr+GFP cells, 169 shILK+GFP cells, 154 shILK+GFP–ILK cells, 162 shILK+I427E cells, 143 shILK+K423D cells, 137 shILK+K426D cells, 125 shILK+I413D cells; *, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey's correction for multiple comparisons (P≤0.01); ns, statistically not significant; P>0.05. (E) Immunoblotting of shScr and shK2 HeLa cells to show protein levels of kindlin-2, ILK, and β-tubulin. (F) Bar graph showing residual kindlin-2 or ILK protein levels in shK2 cells calculated relative to those in shScr cells (mean±s.e.m.); individual data points are indicated (dots; n≥3). (G) Immunofluorescence staining of endogenous vinculin in fixed shScr or shK2 HeLa cells spread on fibronectin-coated glass coverslips and acquired by epifluorescence microscopy. Scale bars: 20 µm. (H) CellProfiler quantification of GFP-positive cells areas pooled across three independent experiments; n=143 shScr+GFP cells, 121 shK2+GFP cells, 145 shK2+GFP-K2 cells, 130 shK2+GFP-K2 LA cells. Data are shown as box and whiskers plot, with whiskers indicating the 10th and 90th percentile range. *, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey's correction for multiple comparisons (P≤0.015); ns, statistically not significant. Microscopy images were linearly and uniformly adjusted for clarity.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Western Blot, Immunofluorescence, Staining, Epifluorescence Microscopy, Microscopy