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

Journal: Frontiers in Pharmacology

Article Title: Agonist-Promoted Phosphorylation and Internalization of the Kappa Opioid Receptor in Mouse Brains: Lack of Connection With Conditioned Place Aversion

doi: 10.3389/fphar.2022.835809

Figure Lengend Snippet: Differential abilities of KOR agonists in promoting KOR internalization in the ventral tegmental areas (VTAs) of male KtdT/KtdT mice. Male KtdT/KtdT mice were injected with saline, Kolliphor EL:ethanol:water (1:1:98) (KEW, vehicle for MOM-SalB), U50,488H (5 mg/kg), MOM-SalB (200 μg/kg), nalfurafine (30 μg/kg), or 42b (5 mg/kg), and 30 min later perfused transcardially with 4% paraformaldehyde. (A,B) KOR-tdT and tyrosine hydroxylase (TH) colocalized in some VTA neurons, and treatment with U50,488, MOM-SalB, and 42b induced KOR internalization VTA, but nalfurafine did not. Coronal sections of frozen brains were obtained and processed for double immunohistochemistry (IHC) with rabbit antibodies against rabbit anti-red fluorescent protein (RFP) (red) and mouse antibodies against tyrosine hydroxylase (TH) (green). Sections were also stained with DAPI for nuclei (blue). (A) Epifluorescence microscopy images of the VTA. Scale bar = 200 μm. (B) A layer of confocal microscopy image. Scale bar = 20 μm. Note that many neurons express both TH and KtdT. Arrows point to internalized receptors, whereas arrow heads indicate receptors on membranes. The experiment was performed on three mice for each group (C–E) Quantitation of agonist-induced KOR internalization. (C) IHC was performed on coronal sections containing the VTA with antibodies against tdT (red) and S6 (cyan), a ribosomal protein in cytosol for identification of all the cells. Sections were also stained with DAPI for nuclei (blue). (D) KEW did not cause KOR internalization, like saline. KEW is the vehicle we used for MOM-SalB. (E) Quantitation of KOR internalization was performed on three sections/mouse at different rostral–caudal positions. Fifteen to 20 neurons per section and three focal planes per neuron from image stacks were measured. Thus, on the average, about 50 neurons/mouse were quantified for internalization as described in the Methods section. Experiments were performed on three mice/group. *** p < 0.001, compared with saline; @@@ p < 0.001, compared with nalfurafine, by one-way ANOVA followed by Tukey’s multiple comparisons test. Each value is mean ± SEM ( n = 3 mice).

Article Snippet: Rabbit anti-red fluorescent protein (RFP) antibody was purchased from Rockland (catalog no. 600-401-379, Limerick, PA, United States).

Techniques: Injection, Saline, Immunohistochemistry, Staining, Epifluorescence Microscopy, Confocal Microscopy, Quantitation Assay

Journal: Frontiers in Plant Science

Article Title: The Intergenic Interplay between Aldose 1-Epimerase-Like Protein and Pectin Methylesterase in Abiotic and Biotic Stress Control

doi: 10.3389/fpls.2017.01646

Figure Lengend Snippet: NbAELP is capable of influencing nucleocytoplasmic traffic. (A) Visualization of GFP expression in epidermal cells of leaves co-agroinjected with GFP:NLS pTα (left) with a projection of several confocal sections superimposed on a bright field image of the same cell (right). Bars = 20 μm. (B) Quantification of GFP:NLS pTα -directed GFP subcellular localization in the plant leaves after co-agroinjection with 35S- NbAELP or vector control. (C) GFP fluorescent epidermal cell cluster under epifluorescence microscopy at 24 h after agroinjection with 35S-GFP:NLS pTα alone (left) or in combination with 35S- NbAELP (right). (D) Quantification of the cell clusters shown in (C) . At least 500 cell clusters were counted for each experiment. The data represent five independent experiments. The standard error bars and P -value for the statistical significance of the difference between the vector control and 35S- NbAELP -injected leaves are indicated.

Article Snippet: For GFP, NbAELP, FLAG or TMV MP detection, the membranes were probed with corresponding antibodies: goat anti-GFP antibodies conjugated with horseradish peroxidase (Rockland Immunochemicals), mouse anti-FLAG monoclonal antibodies (Sigma), mouse polyclonal antibodies against recombinant NbAELP or TMV MP.

Techniques: Expressing, Plasmid Preparation, Control, Epifluorescence Microscopy, Injection

Journal: Frontiers in Plant Science

Article Title: The Intergenic Interplay between Aldose 1-Epimerase-Like Protein and Pectin Methylesterase in Abiotic and Biotic Stress Control

doi: 10.3389/fpls.2017.01646

Figure Lengend Snippet: NbAELP inhibits the accumulation of proNtPME-directed mRNAs. (A) Schematic representation of proNbAELP-based GFP encoding binary vector. LB and RB indicate the left and right T-DNA borders, respectively; 35S, 35S CaMV promoter; term, the 35S terminator of transcription. (B) GFP mRNA content revealed through qRT-PCR (top), and Western blot analysis of the total soluble proteins using anti-GFP antibodies (bottom) in N. benthamiana leaves at 3 days after simultaneous agroinjection of proNtPME- GFP and 35S- NbAELP . The results of densitometry analysis, in relative units, and the standard errors are indicated for the Western blot. The data represent five independent experiments. The P -values were used to assess the statistical significance of the differences in the GFP bands and the GFP mRNA levels. (C) Schematic representation of proNbAELP-based GUS gene encoding binary vector. (D,E) GUS mRNA content revealed through qRT-PCR (D) , and the GUS activity measured in relative light units (E) in N. benthamiana leaves at 3 days after simultaneous agroinjection of proNtPME- GUS and 35S- NbAELP . The P -values were used to assess the statistical significance of the differences in the GUS mRNA levels or GUS activity; n.s., non-significant. (F) Schematic representation of the PME-AELP relationship in the Nicotiana plants.

Article Snippet: For GFP, NbAELP, FLAG or TMV MP detection, the membranes were probed with corresponding antibodies: goat anti-GFP antibodies conjugated with horseradish peroxidase (Rockland Immunochemicals), mouse anti-FLAG monoclonal antibodies (Sigma), mouse polyclonal antibodies against recombinant NbAELP or TMV MP.

Techniques: Plasmid Preparation, Quantitative RT-PCR, Western Blot, Activity Assay