Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig.1. Detection of PYK2 in the oocyte plasma membrane; effect of fertilization. Plasma membrane fractions prepared at 7.5 min post-fertilization were analyzed by western blot and probed with an antibody to the mammalian PYK2 protein, anti-PY402, anti-PY579, or with control rabbit IgG as described in ‘Materials and Methods’ (panel A). To detect changes in PYK2 phosphorylation after fertilization, plasma membrane samples prepared before (UF) and at 1, 2.5, and 10 min post-insemination (m.p.i.), were probed with the anti-PYK2 protein antibody and with the anti-PY579 antibody (panel B) to establish whether the amount or activation state of PYK2 associated with the plasma membrane changed in response to fertilization. Band intensity (‘Materials and Methods’) of the PYK2 PY579 labeled material in panel B was normalized to the PYK2 labeled bands in samples from five membrane preparations and is presented as a bar graph in panel C þ/ S.E.M.

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Clinical Proteomics, Membrane, Western Blot, Control, Phospho-proteomics, Activation Assay, Labeling

Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig. 2. Localization of activated PYK2 in the fertilized oocyte. Oocytes were fixed before and 2.5, 4.5, and 10 m.p.i., then processed for immunofluorescence, labeled with anti-PYK2-PY579 (upper panels) which would detect the activated form of PYK2 or with anti-PYK2 peptide (lower panels) which would detect the total pool of PYK2 protein. Bound antibody was detected with alexa 488-coupled secondary antibody (green) as described in ‘‘Materials and Methods’’. Since the chorion formed a barrier to antibody diffusion, it was punctured multiple times with a micropipette after fixation or removed by dissection in samples fixed after it was fully elevated (10 m.p.i. and later). Magnification is indicated by the bar which represents 100 mm.

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Labeling, Diffusion-based Assay, Dissection

Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig. 3. PYK2 activation co-localized with actin cytoskeletal reorganization. Oocytes were fixed at 1.0 (panels A–C) and 2.5 m.p.i.(panels D–F), then labeled with alexa 565—phalloidin (red) and anti-PYK2-PY579 followed by alexa 488-goat anti-rabbit IgG (green). Confocal images are displayed showing actin in the red channel (A and D), phosphorylated PYK2 in the green channel (B and E), or the merged red and green channels (C and F). Magnification is indicated by the bar which represents 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Activation Assay, Labeling

Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig. 4. PYK2 activation during cleavage and epiboly. Developing embryos were collected at 55, 120, and 240 m.p.i., then processed for confocal immunofluorescence with anti-PYK2 PY579 as in Fig. 2. Magnification is indicated by the bar which represents 100 mm.

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Activation Assay

Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig. 5. Effect of changes in intracellular calcium on PYK2 activation state in the zebrafish oocyte. In order to establish whether oocyte PYK2 could be activated by artificially increasing intracellular calcium levels Oocytes were injected with injection buffer only as a control (A) or with injection buffer containing 25 mM IP3 (B) to artificially drive an increase in intracellular free calcium levels. The control and IP3-injected oocytes were maintained in Hanks-BSA for 4.5 min then fixed. In order to establish whether prevention of the fertilization-induced calcium transient would block PYK2 activation in response to fertilization, oocytes were injected with calcium clamping buffer (C) then fertilized by a mixture of sperm and aquarium water, followed by fixation at 4.5 m.p.i. Oocytes were then prepared for immunofluorescence detection of activated PYK2 with anti-PYK2-PY579

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Activation Assay, Injection, Control, Blocking Assay

Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig. 6. Effect of PYK2 inhibitors on fertilization. Oocytes were treated with AG17, PF04594755, AG82 (control) or DMSO (solvent control) for 45 min, then washed with Hank’s- BSA. Other groups were injected with different concentrations of GST-PERM or GST fusion proteins. The samples were fertilized, then fixed and stained with DAPI at 15 m.p.i. to assess sperm incorporation by the presence of male and female pronuclei, and at 90 m.p.i. to assess cleavage by the presence of a cleavage furrow and two somatic nuclei. An example of zygotes derived from control (DMSO) treated oocytes demonstrating the presence of two pronuclei (A, arrows) and a polar body (pb) demonstrates the criteria for sperm penetration. Panel B demonstrates the presence of a cleavage furrow and two somatic nuclei shown (B, arrows) during telophase of the second mitotic division indicating that fertilization was successful. The % of oocytes that successfully incorporated sperm (C and D, left) or cleaved to the 2-cell stage (C and D, right) are presented as the mean of 4 three experiments þ/ S.E.M. Values represent the mean of three or more experiments þ/ S.E.M. (*)¼significantly different from control (Po0.05).

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Control, Solvent, Injection, Staining, Derivative Assay

Journal: Developmental biology

Article Title: PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

doi: 10.1016/j.ydbio.2012.10.015

Figure Lengend Snippet: Fig. 7. Effect of PYK2 inhibitors on actin reorganization post-fertilization. Oocytes injected with GST-PERM (2.5 mM final) were fertilized and fixed at 2.5 and 4 m.p.i. The distribution of actin was detected with alexa-565-phalloidin (red). Arrows indicate the position of the defective fertilization cone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: A GST fusion protein encoding the N-terminal ERM domain of human PYK2 (PERM domain) was prepared by PCR amplification from a kinase inactive human PYK2 plasmid (Origene catalog #SC323519).

Techniques: Injection

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 1. PYK2 recruitment at the site of sperm-oocyte contact. Zona-free oocytes were incubated with a limiting concentration of sperm and samples were fixed at 30 (A, A′), 45 (B, C), and 60 (D) m.p.i., then processed for confocal immunofluorescence. The distribution of anti-PYK2 protein (green) is shown in the top panels, while f-actin detected by alexa 568- phalloidin (red) is shown in the middle row. Sperm chromatin was detected with DRAQ5 (blue) and the combined images containing all three channels are displayed in the bottom row. Specificity of the anit-PYK2 antibody is seen in column (E) where a pyk2 -/- oocyte collected at 45 m.p.i. was labeled under identical conditions. Magnification is indicated by the bar, which represents 5 µm.

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Incubation, Concentration Assay, Labeling

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 2. PYK2 activation at the site of sperm-oocyte contact. Zona-free oocytes incubated with sperm for 30 min and processed for immunofluorescence as above, were labeled with anti PYK2 PY579 (green), alexa 568-phalloidin (red), and DRAQ5 (blue). The position of an aggregation of phosphorylated (activated) PYK2 and f-actin in close proximity to one of the bound sperm is indicated by the arrows. Magnification is indicated by the bar, which represents 10 µm.

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Activation Assay, Incubation, Labeling

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 4. PYK2 recruitment to sperm binding sites does not require sperm – oocyte fusion. Zona-free CF-1 oocytes were pre-loaded with DRAQ5, then washed and incubated in KSOMaa +15 mg/ml BSA at pH 6.1 or at pH 7.3, as indicated under the X-axis. Capacitated sperm were added and samples were fixed at 20, 30, and 40 min post- insemination. For dye diffusion analysis, groups of 10 oocytes collected at each time point were fixed in formaldehyde to maintain membrane integrity and immediately imaged by confocal microscopy with the 633 nm laser to identify sperm heads that had accumulated DRAQ5 from the oocyte. Duplicate samples for immunofluorescence detection of PYK2 were fixed in 2% formaldehyde with picric acid, and processed through immunolabeling of PYK2. The percent of oocytes that had fused with at least one sperm is indicated by white bars. The percent of oocytes that exhibited PYK2 foci in close proximity to bound sperm is indicated by grey bars. Values along the Y axis represent the mean of 4 experiments ± SEM.

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Binding Assay, Incubation, Membrane, Confocal Microscopy, Immunolabeling

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 5. Recruitment of PYK2 at sperm binding sites of oocytes cultured at pH 6.1. Zona-free oocytes were pre-loaded with DRAQ5, then washed and incubated in KSOMaa +15 mg/ml BSA at pH 6.1, as in Fig. 3. Capacitated sperm were added and samples were fixed with 2% formaldehyde containing picric acid at 20 (A), 30 (B), and 40 (C) minutes post-insemination, then prepared for immunofluorescence detection of PYK2. PYK2 protein detected by alexa 488-anti-rabbit IgG is represented in the green channel (top row), while f-actin detected by alexa 568-phalloidin is seen in the red channel (middle row). Sperm chromatin labeled with DRAQ5 is seen in the blue channel. Sperm binding sites where PYK2 accumulation was observed are indicated by the arrows. Magnification is indicated by the bar, which represents 10 µm.

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Binding Assay, Cell Culture, Incubation, Labeling

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 6. Role of PYK2 in gamete fusion and sperm incorporation. The timing and extent of sperm-oocyte fusion was quantified in WT (white bars) and pyk2 -/- (grey bars) oocytes pre-loaded with DRAQ5 as in Fig. 3. Samples of oocytes were collected at 60, 90, and 120 m.p.i. to correlate with the sperm incorporation time course (below). Sperm heads bound to the oocyte surface that accumulated DRAQ5 from the oocyte were considered to have fused with the oocyte plasma membrane (panel A). Values represent the mean of 4 experiments including 116 WT oocytes and 126 pyk2 -/- oocytes. The timing and extent of sperm incorporation was quantified in separate experiments (Panel B) where WT (white bars) and pyk2 -/- (grey bars) oocytes were fixed and labeled with DRAQ 5 to label all sperm heads and with alexa 568-phalloidin to label the cortical actin layer. Sperm heads that were located within the oocyte cytoplasm and which showed evidence of nuclear decondensation were considered to be incorporated. Values in panel B represent the mean of 5 groups including 123 WT oocytes and 133 pyk2 -/- oocytes ± SEM. (*) t- test indicated that the WT and pyk2 -/- means were significantly different (P=0.01).

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Clinical Proteomics, Membrane, Labeling

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 7. Role of PYK2 in actin polymerization at sperm-oocyte binding sites. Oocytes in which PYK2 was suppressed by different methods were incubated with capacitated sperm, then fixed at 45 m.p.i. to determine whether they could respond to sperm binding by accumulation of f-actin in the cortical actin layer. The distribution of actin at sites of sperm contact was detected by labeling with alexa 568-phalloidin to detect f-actin (red) and DRAQ5 to detect sperm DNA (blue). PYK2 activity was suppressed in WT oocytes by three independent methods. The effect of chemical inhibition is shown in panels A and B, where oocytes were treated with 0.1% DMSO as a solvent control (A) or with the inhibitor PF0454799 at 10uM for 30 min, then washed prior to addition of sperm (B). The effect of the dominant-negative PERM fusion construct was tested by injecting oocytes with cRNA encoding eGFP as a control (C), or with cRNA encoding the inhibitory PERM-eGFP construct (D). Panels E and F demonstrate the effect of pyk2 knockout on the response of oocytes to bound sperm with examples of sperm bound to a WT oocyte (E) and a pyk2 -/- oocyte (F). Magnification is indicated by the bar which represents 5 µm.

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Binding Assay, Incubation, Labeling, Activity Assay, Inhibition, Solvent, Control, Dominant Negative Mutation, Construct, Knock-Out

Journal: Developmental biology

Article Title: Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

doi: 10.1016/j.ydbio.2017.05.016

Figure Lengend Snippet: Fig. 8. PYK2 is required for enhanced actin polymerization at a subset of sperm-oocyte binding sites. Zona-free oocytes collected from WT and PYK2 -/- females were incubated with capacitated sperm for 45 min as described in Section 2 then fixed and labeled with alexa 568-phalloidin to detect f-actin (red) and DRAQ5 to detect sperm DNA (blue). The distribution of bound sperm and f-actin in the oocyte cortex was recorded by Z-stack confocal imaging and quantified by linescan analysis as described in Section 2. Panel A demonstrates the distribution of sperm heads (labeled S31,33, 34, 35 and 36) detected by DRAQ5 fluorescence in the blue channel (Y axis, upper trace) around the perimeter of a representative optical section through a WT oocyte. The corresponding alexa 568-phalloidin fluorescence is expressed in the red channel (Y axis, lower trace). Instances where sperm binding sites are associated with increased alexa 568-fluorescence are indicated by arrows. “actin cap” represents the thickening of the cortical actin layer that normally occurs over the MII spindle. Panel B demonstrated the relative integrated alexa 568-phalloidin fluorescence (Y axis) calculated for each sperm binding site (n=123) from 7 WT oocytes and (n=134) from 7 pyk2 -/- oocytes. “0” representing no change relative to the adjacent cortex and positive values representing an increase in fluorescence intensity relative to the adjacent cortex. Panels C and D display the number of sperm binding sites associated with given levels of relative integrated alexa 568-phalloidin fluorescence (X axis).

Article Snippet: An eGFP-linked dominant-negative construct encoding the N-terminal ERM domain (aa1-370) of PYK2 was amplified from the human PYK2 transcript variant 1 cDNA (Origene, Rockville, MD) using the following primers: (forward; CCCAAGCTTATGTCTGGGGTGTCCGAGCCC); (reverse; CCCAAGCTTGCTGTTCCGCTTCTCACCATCTT) which contained a Hind III site used to ligate the product into the N-terminal cloning site of pEGFPN1 (Clontech, Mountain View, CA).

Techniques: Binding Assay, Incubation, Labeling, Imaging

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: Hypoxia induces cell membranous interaction between Pyk2 and NOX5. a KYSE30 and KYSE410 cells were cultured under normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Immunocomplexes were then immunoblotted using NOX5 and Pyk2 antibodies. The efficacy of membrane protein extraction was examined using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) in cell membrane lysis. b KYSE30 and KYSE410 cells were cultured under normoxic or hypoxic condition for 1 h. Intracellular Ca 2+ level was evaluated using Calcium detection assay kit. c KYSE30 and KYSE410 cells were pretreated with 10 μM Ca 2+ chelator-BAPTA-AM for 30 min and then exposed to normoxic or hypoxic condition for 1 h. The Pyk2 (Tyr 402 ) phosphorylation was assayed by Pyk2 activation ELISA assay. d , e KYSE30 control, NOX5, or Pyk2-ovexexpressing cells were pretreated with 10 μM Ca 2+ chelator-BAPTA-AM for 30 min or control solvent ( d ), or transfected with control vector or Pyk2 Y402F plasmid ( e ), were cultured normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Immunocomplexes were then immunoblotted using NOX5 and Pyk2 antibodies. The efficacy of membrane protein extraction was evaluated using immunoblotting to assess the expression of α1-ATPase (membrane biomarker) in cell membrane lysis. f The KYSE30 and KYSE410 control shRNA or NOX5 shRNA cells were exposed to hypoxia for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Then, the Pyk2 complex-produced H 2 O 2 was examined using an Amplex red hydrogen peroxide assay kit. g KYSE30 control or NOX5-overexpressing cells pretreated with or without 10 μM Ca 2+ chelator-BAPTA-AM, or transfected with control vector or Pyk2 Y402F plasmid, were exposed to normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Then, the Pyk2 complex-produced H 2 O 2 was assayed using an Amplex red hydrogen peroxide assay kit. *** P < 0.001; two-tailed unpaired Student’s t -test. Error bars represent mean ± SD of five independent experiments

Article Snippet: Levels of activated Src and Pyk2 in the cell lysis (~20 μg protein per sample) were measured using the human phosphor-Src (Tyr 419 ) ELISA kit (Raybiotech; catalog# PEL-SRC-Y419-T) and the human phosphor-Pyk2 (Tyr 402 ) ELISA kit (Raybiotech; catalog# PEL-PYK2-Y402-T) following the manufacturer’s protocols.

Techniques: Cell Culture, Immunoprecipitation, Protein Extraction, Western Blot, Expressing, Biomarker Assay, Lysis, Detection Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, shRNA, Produced, Amplex Red Hydrogen Peroxide Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: Pyk2 recruits c-Abl to enhance NOX5 activity in Pyk2/NOX5 complex. a KYSE30 or KYSE410 cells were transfected with vector, Flag-Pyk2 wild type (wt), or Flag- Pyk2 Y881F mutant plasmid and then cultured under normoxia or hypoxia for 1 h. Cell membrane lysates were immunoprecipitated with c-Abl antibody. Immunocomplexes were then immunoblotted using NOX5 and c-Abl antibodies ( a ). Membranous Src was immunoprecipitated with an anti-Src antibody. Oxidized Src levels were measured using a modified OxyBlot protein detection kit ( a ). The efficacy of membrane protein extraction was evaluated using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) ( a ) in cell membrane lysis. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. b–d HeLa cells were co-transfected HA-c-Abl wt or kinase-dead (KD) K290R mutant with Flag-tagged NOX5 wt, Y476/478F, Y487F, or Y519F plasmid, respectively. Cell lysates were immunoprecipitated with the antibody against Flag. Cell membrane lysates were then immunoblotted using antibodies against flag and phosphotyrosine. Membranous Src was immunoprecipitated with an anti-Src antibody. Oxidized Src levels were assayed using a modified OxyBlot protein detection kit ( b ). The transfection efficacy was measured using immunoblotting ( c ). The Pyk2 complex-derived H 2 O 2 was tested using an Amplex red hydrogen peroxide assay kit ( d ). e , f The indicated ESCC cells were transfected with control vector, Flag-NOX5 wt, or Flag-NOX5 Y476/478F (mutant) plasmid, respectively, and then cultured under normoxic or hypoxic condition for 1 h. Oxidized Src levels were measured using a modified OxyBlot protein detection kit ( e ). The phosphorylation of Src Tyr 419 in Pyk2 complex was immunoprecipitated with the antibody against Pyk2 and then immunoblotted using antibodies against Pyk2 and pSrc (Tyr 419 ) ( e ). The efficacy of membrane protein extraction was evaluated using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) ( e ) in cell membrane lysis. The Pyk2 complex-produced H 2 O 2 was assayed using an Amplex red hydrogen peroxide assay kit ( f ). *** P < 0.001; two-tailed unpaired Student’s t -test. Error bars represent mean ± SD of five independent experiments

Article Snippet: Levels of activated Src and Pyk2 in the cell lysis (~20 μg protein per sample) were measured using the human phosphor-Src (Tyr 419 ) ELISA kit (Raybiotech; catalog# PEL-SRC-Y419-T) and the human phosphor-Pyk2 (Tyr 402 ) ELISA kit (Raybiotech; catalog# PEL-PYK2-Y402-T) following the manufacturer’s protocols.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Cell Culture, Immunoprecipitation, Modification, Protein Extraction, Western Blot, Expressing, Biomarker Assay, Lysis, Derivative Assay, Amplex Red Hydrogen Peroxide Assay, Produced, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: Proposed Model of Membranous NOX5-derived local ROS oxidizes and activates Src to promote growth and invasion of ESCC cells. Hypoxia stimulated the interaction between NOX5 and Pyk2 on cell membrane via enhancing intracellular Ca 2+ -mediated Pyk2 Tyr 402 phosphorylation. Subsequently, Pyk2 acted as a scaffold via its Tyr 881 site for c-Abl phosphorylating the catalytic domain of NOX5 Tyr 476/478 and then upregulated H 2 O 2 inside the Pyk2/NOX5 complex to oxidize and activate Src

Article Snippet: Levels of activated Src and Pyk2 in the cell lysis (~20 μg protein per sample) were measured using the human phosphor-Src (Tyr 419 ) ELISA kit (Raybiotech; catalog# PEL-SRC-Y419-T) and the human phosphor-Pyk2 (Tyr 402 ) ELISA kit (Raybiotech; catalog# PEL-PYK2-Y402-T) following the manufacturer’s protocols.

Techniques: Derivative Assay