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R&D Systems human cathepsin b duoset elisa dy2176 pyk2 inhibitor pf 431396

Fig. 7 Galectin-3 expression induces activation of <t>PYK2,</t> STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Proﬁler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantiﬁed and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantiﬁed and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Human Cathepsin B Duoset Elisa Dy2176 Pyk2 Inhibitor Pf 431396, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 18 article reviews

human cathepsin b duoset elisa dy2176 pyk2 inhibitor pf 431396 - by Bioz Stars, 2026-06

94/100 stars

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1) Product Images from "Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells."

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

Journal: Cell death & disease

doi: 10.1038/s41419-023-05789-x

Fig. 7 Galectin-3 expression induces activation of PYK2, STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Proﬁler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantiﬁed and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantiﬁed and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Figure Legend Snippet: Fig. 7 Galectin-3 expression induces activation of PYK2, STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Proﬁler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantiﬁed and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantiﬁed and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Techniques Used: Expressing, Activation Assay, Comparison, Control, Phospho-proteomics, Western Blot

Fig. 8 Galectin-3 induces CTSB secretion through PYK2-GSK3α/β activation. SW620 (A) and HCT116 (B) cells were treated with 10 µg/ml galectin-3 or BSA without or with SB216763 (SB), PF431396 (PF) or DMSO overnight. The concentrations of cathepsin-B in the supernatants were analysed by ELISA. Data are presented as mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Figure Legend Snippet: Fig. 8 Galectin-3 induces CTSB secretion through PYK2-GSK3α/β activation. SW620 (A) and HCT116 (B) cells were treated with 10 µg/ml galectin-3 or BSA without or with SB216763 (SB), PF431396 (PF) or DMSO overnight. The concentrations of cathepsin-B in the supernatants were analysed by ELISA. Data are presented as mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

Image Search Results

a , b DIO wild-type mice that have been on HFD diet were implanted a guide cannula into the lateral ventricle. Mice were treated with vehicle or PF centrally (icv), and vehicle or TubA (15 mg/kg) was administered peripherally once daily. The daily weight change curves ( a ) and cumulative weight change at the end of the treatment ( b ) ( n = 4 Veh/PF mice; n = 7 Veh/PF + TubA mice). c Growth curves and d average food intake of wild-type or Pyk2 KO mice on HFD. Body weight change of wild-type and Pyk2 KO DIO mice during ( e ) and at the end of ( f ) daily TubA treatments. Average food intake of the animals during vehicle acclimation ( g ) and TubA treatments ( h ). i Cumulative food intake of the wild-type and Pyk2 KO mice at the end of TubA treatments. j Blood glucose of the mice before and after TubA treatments. k Difference in the blood glucose of the mice following TubA treatment ( n = 6 WT mice; n = 5 Pyk2 KO mice). Data shown are representative of experiments conducted once ( a – d ) or twice ( e – k ) in separate cohorts of animals with similar results. Data are represented as mean ± SEM and analyzed by t -test ( d , f – i , k ), mixed effect analysis ( a ), one-way ANOVA ( b ), two-way ANOVA ( c , e , j ).

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a , b DIO wild-type mice that have been on HFD diet were implanted a guide cannula into the lateral ventricle. Mice were treated with vehicle or PF centrally (icv), and vehicle or TubA (15 mg/kg) was administered peripherally once daily. The daily weight change curves ( a ) and cumulative weight change at the end of the treatment ( b ) ( n = 4 Veh/PF mice; n = 7 Veh/PF + TubA mice). c Growth curves and d average food intake of wild-type or Pyk2 KO mice on HFD. Body weight change of wild-type and Pyk2 KO DIO mice during ( e ) and at the end of ( f ) daily TubA treatments. Average food intake of the animals during vehicle acclimation ( g ) and TubA treatments ( h ). i Cumulative food intake of the wild-type and Pyk2 KO mice at the end of TubA treatments. j Blood glucose of the mice before and after TubA treatments. k Difference in the blood glucose of the mice following TubA treatment ( n = 6 WT mice; n = 5 Pyk2 KO mice). Data shown are representative of experiments conducted once ( a – d ) or twice ( e – k ) in separate cohorts of animals with similar results. Data are represented as mean ± SEM and analyzed by t -test ( d , f – i , k ), mixed effect analysis ( a ), one-way ANOVA ( b ), two-way ANOVA ( c , e , j ).

Article Snippet: The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD).

Techniques:

$HEK-293T cells were transiently transfected with 0.25 µg Stat3 (lanes 2–10) and increasing amounts (0.25–1.75 μg) of wild-type or mutant Fak (FAK Y397F ) ( a ) or wild-type or kinase-dead (KD) Pyk2 ( b ). The FAK Y397F mutation prevents phosphorylation at Tyr 397 , abolishing kinase activity ( a , lanes 7–10). Total cell lysates (TCL) were immunoblotted with the indicated antibodies. The migration difference between wild-type and Pyk2 KD reflects the GFP tag in the KD construct. c STAT3, PYK2, or FAK were transiently overexpressed in HEK-293T cells as indicated. PYK2 or FAK was immunoprecipitated, and TCL or the immunoprecipitated fractions were analyzed by western blot using the indicated antibodies. d Leptin enhances the interaction of hypothalamic focal adhesion kinases with STAT3. Lean wild-type mice received vehicle or leptin (5 mg/kg). Hypothalamic were collected 30 min later, crosslinked (10% formalin), homogenized, and immunoprecipitated with STAT3 or IgG control antibodies. Immunoprecipitates were analyzed for the indicated proteins. Fak and Pyk2 mRNA ( e ) and protein ( f ) expression in N1 cells stably expressing shScr, shFak, or shPyk2 constructs. g LepRb-expressing N1 cells with shScr, shFak, or shPyk2 were stimulated with leptin (40 ng/mL), and pSTAT3 and total STAT3 were analyzed at the indicated times. h Quantification of pSTAT3/STAT3 signals; inset shows the area under the curve. i N1 cells transfected with LepRb and either shScr or shRNAs targeting both Fak and Pyk2 were stimulated with 40 ng/mL leptin. Total lysates were analyzed by immunoblot for pSTAT3 and total STAT3. j N1 cells stably expressing LepRb and a STAT3-driven luciferase reporter were treated with PF and leptin. Luminescence was measured 24 h later. k Relative leptin-induced luminescence at 0, 2, and 128 ng/mL leptin with PF concentrations of 0 (Veh), 330 nM, 1 µM, or 3.3 µM. Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Data shown are representative of an experiment conducted once ( a – f ) or twice ( g – k ) with similar results. Data are represented as mean ± SEM and analyzed by one-way ( h ) or two-way ANOVA ( e , k ).$

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: HEK-293T cells were transiently transfected with 0.25 µg Stat3 (lanes 2–10) and increasing amounts (0.25–1.75 μg) of wild-type or mutant Fak (FAK Y397F ) ( a ) or wild-type or kinase-dead (KD) Pyk2 ( b ). The FAK Y397F mutation prevents phosphorylation at Tyr 397 , abolishing kinase activity ( a , lanes 7–10). Total cell lysates (TCL) were immunoblotted with the indicated antibodies. The migration difference between wild-type and Pyk2 KD reflects the GFP tag in the KD construct. c STAT3, PYK2, or FAK were transiently overexpressed in HEK-293T cells as indicated. PYK2 or FAK was immunoprecipitated, and TCL or the immunoprecipitated fractions were analyzed by western blot using the indicated antibodies. d Leptin enhances the interaction of hypothalamic focal adhesion kinases with STAT3. Lean wild-type mice received vehicle or leptin (5 mg/kg). Hypothalamic were collected 30 min later, crosslinked (10% formalin), homogenized, and immunoprecipitated with STAT3 or IgG control antibodies. Immunoprecipitates were analyzed for the indicated proteins. Fak and Pyk2 mRNA ( e ) and protein ( f ) expression in N1 cells stably expressing shScr, shFak, or shPyk2 constructs. g LepRb-expressing N1 cells with shScr, shFak, or shPyk2 were stimulated with leptin (40 ng/mL), and pSTAT3 and total STAT3 were analyzed at the indicated times. h Quantification of pSTAT3/STAT3 signals; inset shows the area under the curve. i N1 cells transfected with LepRb and either shScr or shRNAs targeting both Fak and Pyk2 were stimulated with 40 ng/mL leptin. Total lysates were analyzed by immunoblot for pSTAT3 and total STAT3. j N1 cells stably expressing LepRb and a STAT3-driven luciferase reporter were treated with PF and leptin. Luminescence was measured 24 h later. k Relative leptin-induced luminescence at 0, 2, and 128 ng/mL leptin with PF concentrations of 0 (Veh), 330 nM, 1 µM, or 3.3 µM. Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Data shown are representative of an experiment conducted once ( a – f ) or twice ( g – k ) with similar results. Data are represented as mean ± SEM and analyzed by one-way ( h ) or two-way ANOVA ( e , k ).

Article Snippet: The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD).

Techniques: Transfection, Mutagenesis, Phospho-proteomics, Activity Assay, Migration, Construct, Immunoprecipitation, Western Blot, Control, Expressing, Stable Transfection, Luciferase, Imaging

a – k Hypothalamic sections from lean wild-type mice, ad libitum fed, overnight fasted, or overnight fasted and leptin (3 h) treated (ip) were stained with fluorescent RNAscope probes targeting LepR , Fak or Pyk2 transcripts. a The percent of LepR + cells with Fak and Pyk2 in the ARC, VMH, and DMH. b Representative images of hypothalamic sections covering ARC, VMH, and DMH for LepR , Fak , Pyk2 expressions. The scale bar represents 100 μm. Representative RNAscope images and region specific and LepR + specific transcript quantification in the VMH ( c – e ), DMH ( f – h ), and ARC ( i – k ) ( n = 5 mice per group). Red: LepR , yellow: Pyk2 , green: Fak , blue: DAPI. Arrowheads indicate triple colocalization of LepR , Pyk2 , and Fak . The scale bar represents 50 μm ( c ). The transcripts in the LepR + cells were expressed as the counted pixels within the LepR + cells, except for Pyk2 signal in the DMH, which is expressed as the total fluorescence due to high signal intensity. Data shown are representative of an experiment conducted once. Data are represented as mean ± SEM and are analyzed by one-way ANOVA ( d , e , g , h , j , k ).

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a – k Hypothalamic sections from lean wild-type mice, ad libitum fed, overnight fasted, or overnight fasted and leptin (3 h) treated (ip) were stained with fluorescent RNAscope probes targeting LepR , Fak or Pyk2 transcripts. a The percent of LepR + cells with Fak and Pyk2 in the ARC, VMH, and DMH. b Representative images of hypothalamic sections covering ARC, VMH, and DMH for LepR , Fak , Pyk2 expressions. The scale bar represents 100 μm. Representative RNAscope images and region specific and LepR + specific transcript quantification in the VMH ( c – e ), DMH ( f – h ), and ARC ( i – k ) ( n = 5 mice per group). Red: LepR , yellow: Pyk2 , green: Fak , blue: DAPI. Arrowheads indicate triple colocalization of LepR , Pyk2 , and Fak . The scale bar represents 50 μm ( c ). The transcripts in the LepR + cells were expressed as the counted pixels within the LepR + cells, except for Pyk2 signal in the DMH, which is expressed as the total fluorescence due to high signal intensity. Data shown are representative of an experiment conducted once. Data are represented as mean ± SEM and are analyzed by one-way ANOVA ( d , e , g , h , j , k ).

Article Snippet: The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD).

Techniques: Staining, RNAscope, Fluorescence

a – c Fasted lean wild-type and Pyk2 KO mice were treated (ip) with vehicle or leptin, and food intake and body weights were followed for 24 h. a Time course of food intake, b food intake presented as normalized to the respective vehicle groups, and c weight change of the animals ( a – c , n = 6 mice per group). d , e Hypothalamic STAT3 phosphorylation was analyzed 30, 60, and 120 min post leptin injection in lean wild-type and Pyk2 KO mice ( n = 1 Vehicle mouse; n = 3 Leptin mice, per timepoint). Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Data shown are representative of experiments conducted twice in separate cohorts of animals with similar results. Data are represented as mean ± SEM and are analyzed by two-way ANOVA.

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a – c Fasted lean wild-type and Pyk2 KO mice were treated (ip) with vehicle or leptin, and food intake and body weights were followed for 24 h. a Time course of food intake, b food intake presented as normalized to the respective vehicle groups, and c weight change of the animals ( a – c , n = 6 mice per group). d , e Hypothalamic STAT3 phosphorylation was analyzed 30, 60, and 120 min post leptin injection in lean wild-type and Pyk2 KO mice ( n = 1 Vehicle mouse; n = 3 Leptin mice, per timepoint). Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Data shown are representative of experiments conducted twice in separate cohorts of animals with similar results. Data are represented as mean ± SEM and are analyzed by two-way ANOVA.

Article Snippet: The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD).

Techniques: Phospho-proteomics, Injection, Imaging

Lean wild type mice were bilaterally injected with AAV particles encoding shScr or shPyk2/Fak in the mediobasal hypothalamus and DMH. Two weeks later mice were either maintained on regular chow or switched to HFD. a – c Growth curves of the cohorts on the indicated diets expressed either as absolute body weight or percent weight gain. Week 0 indicates 2 weeks post viral injection. d Food intake of the cohorts measured over 24 h around week 3. Body composition of the cohort measured around week 2 using NMR and plotted as absolute tissue mass ( e , g ) or normalized to body weight ( f , h ). i Blood glucose of the mice measured during the daytime after a 4 h fast ( a – i , n = 6 shScr Chow mice; n = 7 shScr HFD mice; n = 9 shFak/Pyk2 mice). Cohorts on chow diet (group caged) were treated with either vehicle or leptin (5 mg/kg, ip) prior to dark cycle and their food intake ( j ) ( n = 8 mice per group) and body weight ( k ) was followed for 24 h ( n = 25 shScr mice; n = 24 shFak/Pyk2 mice). l – o DIO cohorts were treated with vehicle or TubA (15 mg/kg, ip, daily). Absolute weight change ( l ), percent weight change ( m ), average food intake ( n ), and body weights ( o ) are measured daily ( l – o , n = 19 shScr mice; n = 16 shFak/Pyk2 mice). Data shown are representative of experiments conducted twice in separate cohorts of animals with similar results. Data are represented as mean ± SEM and are analyzed by one-way ( d , i ) or two-way ANOVA ( a – c , e – h , j – o ).

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: Lean wild type mice were bilaterally injected with AAV particles encoding shScr or shPyk2/Fak in the mediobasal hypothalamus and DMH. Two weeks later mice were either maintained on regular chow or switched to HFD. a – c Growth curves of the cohorts on the indicated diets expressed either as absolute body weight or percent weight gain. Week 0 indicates 2 weeks post viral injection. d Food intake of the cohorts measured over 24 h around week 3. Body composition of the cohort measured around week 2 using NMR and plotted as absolute tissue mass ( e , g ) or normalized to body weight ( f , h ). i Blood glucose of the mice measured during the daytime after a 4 h fast ( a – i , n = 6 shScr Chow mice; n = 7 shScr HFD mice; n = 9 shFak/Pyk2 mice). Cohorts on chow diet (group caged) were treated with either vehicle or leptin (5 mg/kg, ip) prior to dark cycle and their food intake ( j ) ( n = 8 mice per group) and body weight ( k ) was followed for 24 h ( n = 25 shScr mice; n = 24 shFak/Pyk2 mice). l – o DIO cohorts were treated with vehicle or TubA (15 mg/kg, ip, daily). Absolute weight change ( l ), percent weight change ( m ), average food intake ( n ), and body weights ( o ) are measured daily ( l – o , n = 19 shScr mice; n = 16 shFak/Pyk2 mice). Data shown are representative of experiments conducted twice in separate cohorts of animals with similar results. Data are represented as mean ± SEM and are analyzed by one-way ( d , i ) or two-way ANOVA ( a – c , e – h , j – o ).

Article Snippet: The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD).

Techniques: Injection

Peripheral HDAC6 inhibition induces a systemic signal(s) of unknown nature (depicted as purple triangle) that act as leptin sensitizers leading to weight loss in diet-induced obese mice. Central inhibition or genetic ablation of HDAC6 does not induce weight loss. This cell non-autonomous mechanism involves increased expression of the focal adhesion kinases, FAK and PYK2, which can directly phosphorylate the transcription factor STAT3 acting downstream of leptin receptor. STAT3 is also phosphorylated by its canonical kinase, JAK2, in response to leptin. Phosphorylated STAT3 translocates to the nucleus and induces a transcriptional program leading to suppression of food intake and increased energy expenditure. Created in BioRender. Cakir, I. (2026) https://BioRender.com/jd9puvs .

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: Peripheral HDAC6 inhibition induces a systemic signal(s) of unknown nature (depicted as purple triangle) that act as leptin sensitizers leading to weight loss in diet-induced obese mice. Central inhibition or genetic ablation of HDAC6 does not induce weight loss. This cell non-autonomous mechanism involves increased expression of the focal adhesion kinases, FAK and PYK2, which can directly phosphorylate the transcription factor STAT3 acting downstream of leptin receptor. STAT3 is also phosphorylated by its canonical kinase, JAK2, in response to leptin. Phosphorylated STAT3 translocates to the nucleus and induces a transcriptional program leading to suppression of food intake and increased energy expenditure. Created in BioRender. Cakir, I. (2026) https://BioRender.com/jd9puvs .

Article Snippet: The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD).

Techniques: Inhibition, Expressing

Fig. 6 PYK2 is a potential target of Entrectinib. (A) The binding model between Entrectinib (pink) and the crucial residues of PYK2 (purple). (B) The bind ing affinity between PYK2 and Entrectinib was analyzed by Microscale thermophoresis (MST). (C) Cellular Thermal Shift Assay and Western blot were performed to evaluate the stability of PYK2 after incubation with or without Entrectinib at different temperatures. (D) Drug Affinity Responsive Target Sta bility and Western blot were performed to evaluate the resistance of PYK2 to enzymatic hydrolysis. (E) The phosphorylation levels of PYK2 in cells treated by TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM) were analyzed by western blot. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. ###P < 0.001 versus control group. ****P < 0.0001 versus TGFβ2 group

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 6 PYK2 is a potential target of Entrectinib. (A) The binding model between Entrectinib (pink) and the crucial residues of PYK2 (purple). (B) The bind ing affinity between PYK2 and Entrectinib was analyzed by Microscale thermophoresis (MST). (C) Cellular Thermal Shift Assay and Western blot were performed to evaluate the stability of PYK2 after incubation with or without Entrectinib at different temperatures. (D) Drug Affinity Responsive Target Sta bility and Western blot were performed to evaluate the resistance of PYK2 to enzymatic hydrolysis. (E) The phosphorylation levels of PYK2 in cells treated by TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM) were analyzed by western blot. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. ###P < 0.001 versus control group. ****P < 0.0001 versus TGFβ2 group

Article Snippet: In brief, after dyeing with N-hydroxysuccinimide, the recombinant human PYK2 protein (Solarbio, China) was diluted and mixed with Entrectinib of different concentrations.

Techniques: Binding Assay, Microscale Thermophoresis, Thermal Shift Assay, Western Blot, Incubation, Phospho-proteomics, Control

Fig. 7 The knockdown of PYK2 inhibits the TGFβ2-induced EMT through a non-Smad signaling pathway. (A) The knockdown efficiency of PYK2 was evaluated by western blotting. (B) The knockdown of PYK2 inhibited the EMT induced by TGFβ2. The efficacy of Entrectinib is influenced by PYK2 knock down. (C) PYK2 knockdown inhibited the activation of AKT, JNK, ERK, and P38. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. #P < 0.05 and ##P < 0.01 versus the control group

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 7 The knockdown of PYK2 inhibits the TGFβ2-induced EMT through a non-Smad signaling pathway. (A) The knockdown efficiency of PYK2 was evaluated by western blotting. (B) The knockdown of PYK2 inhibited the EMT induced by TGFβ2. The efficacy of Entrectinib is influenced by PYK2 knock down. (C) PYK2 knockdown inhibited the activation of AKT, JNK, ERK, and P38. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. #P < 0.05 and ##P < 0.01 versus the control group

Article Snippet: In brief, after dyeing with N-hydroxysuccinimide, the recombinant human PYK2 protein (Solarbio, China) was diluted and mixed with Entrectinib of different concentrations.

Techniques: Knockdown, Western Blot, Activation Assay, Control

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Figure Lengend Snippet: Fig. 7 Galectin-3 expression induces activation of PYK2, STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Proﬁler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantiﬁed and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantiﬁed and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Article Snippet: Biotinylated-anti-galectin-3 (BAF1154) antibody and antibodies against galectin-3 (MAB1154), STAT1 (MAB1490), Phospho-STAT1(MAB2894), STAT3 (MAB1799), Phospho-STAT3 (MAB4934), PYK2 (AF4589), phosphorPYK2 (MAB6210), GSK3α/β (AF2157), Phospho-GSK3α/β (AF1590); Proteome Profiler Human Protease Array (ARY021B), Proteome Profiler Human Phospho-Kinase Array (ARY003C); Human MMP-13 DuoSet ELISA (DY511), Human Cathepsin-B DuoSet ELISA (DY2176) PYK2 inhibitor PF-431396 (4278) and GSK3α/β inhibitor SB 216763 (1616/1) were all purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Activation Assay, Comparison, Control, Phospho-proteomics, Western Blot

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Figure Lengend Snippet: Fig. 8 Galectin-3 induces CTSB secretion through PYK2-GSK3α/β activation. SW620 (A) and HCT116 (B) cells were treated with 10 µg/ml galectin-3 or BSA without or with SB216763 (SB), PF431396 (PF) or DMSO overnight. The concentrations of cathepsin-B in the supernatants were analysed by ELISA. Data are presented as mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Techniques: Expressing, Activation Assay, Comparison, Control, Phospho-proteomics, Western Blot

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Phospho-PYK2-Y402 localizes at invadopodia and regulates invadopodia formation and cell invasion in naïve WM793 cells. (A) Representative images of fluorescence staining shows that PYK2-EGFP was localized at invadopodia (actin staining). (B) Representative images of fluorescence staining show p-PYK2 localized at invadopoida. (C–E) Western blot analysis of expressions of PYK2 and p-PYK2 (C), quantification of invadopodia (D) and invaded cells (E) in PYK2-knockdown and control WM793 cells ( n = 3). (F–H) Western blot analyses of PYK2 and p-PYK2 expressions in PYK2-transfected and control WM793 cells (F), and quantification of invadopodia (G) and invaded cells (H) in PYK2-overexpressing and control WM793 cell ( n = 3). (I) Knockdown of PYK2 in 1205Lu cells significantly reduced lung metastases in a mouse model. Scale bars = 10 μm in the main images; Scale bars = 2 μm in the inserts. * P < 0.05; *** P < 0.001.

Journal: Cancer Biology & Medicine

Article Title: PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas

doi: 10.20892/j.issn.2095-3941.2020.0294

Figure Lengend Snippet: Phospho-PYK2-Y402 localizes at invadopodia and regulates invadopodia formation and cell invasion in naïve WM793 cells. (A) Representative images of fluorescence staining shows that PYK2-EGFP was localized at invadopodia (actin staining). (B) Representative images of fluorescence staining show p-PYK2 localized at invadopoida. (C–E) Western blot analysis of expressions of PYK2 and p-PYK2 (C), quantification of invadopodia (D) and invaded cells (E) in PYK2-knockdown and control WM793 cells ( n = 3). (F–H) Western blot analyses of PYK2 and p-PYK2 expressions in PYK2-transfected and control WM793 cells (F), and quantification of invadopodia (G) and invaded cells (H) in PYK2-overexpressing and control WM793 cell ( n = 3). (I) Knockdown of PYK2 in 1205Lu cells significantly reduced lung metastases in a mouse model. Scale bars = 10 μm in the main images; Scale bars = 2 μm in the inserts. * P < 0.05; *** P < 0.001.

Article Snippet: TMAs were immunohistochemically stained with mouse monoclonal anti-human p-PYK2-Y402 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Fluorescence, Staining, Western Blot, Knockdown, Control, Transfection

P-PYK2 mediates vemurafenib-induced invadopodia formation in vemurafenib resistant WM793 cells. (A, B) Western blot analyses (A) and quantification of p-PYK2 levels (B) in naïve and vemurafenib resistant WM793 cells ( n = 3). (C, D) Western blot analyses (C) and the quantification (D) of p-PYK2 levels in WM793 cells treated with and without vemurafenib at the indicated times ( n = 3). (E) Quantification of invadopodia in PYK2-knockdown and control cells treated with and without vemurafenib ( n = 3). *** P < 0.001.

Journal: Cancer Biology & Medicine

Article Title: PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas

doi: 10.20892/j.issn.2095-3941.2020.0294

Figure Lengend Snippet: P-PYK2 mediates vemurafenib-induced invadopodia formation in vemurafenib resistant WM793 cells. (A, B) Western blot analyses (A) and quantification of p-PYK2 levels (B) in naïve and vemurafenib resistant WM793 cells ( n = 3). (C, D) Western blot analyses (C) and the quantification (D) of p-PYK2 levels in WM793 cells treated with and without vemurafenib at the indicated times ( n = 3). (E) Quantification of invadopodia in PYK2-knockdown and control cells treated with and without vemurafenib ( n = 3). *** P < 0.001.

Article Snippet: TMAs were immunohistochemically stained with mouse monoclonal anti-human p-PYK2-Y402 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Knockdown, Control

The PYK2 inhibitor, PF562711, abrogates vemurafenib-induced invadopodia formation and cell invasion. (A–C) Western blot analysis of p-PYK2 levels (A), representative images of fluorescence staining of actin (B), and quantification of invadopodia (C) in WM793 cells treated with dimethyl sulfoxide or PF562711 ( n = 3). (D) Quantification of invadopodia in WM793 cells treated with and without vemurafenib and/or PF562711 ( n = 3). (E, F) Quantification of invadopodia (E) and invaded cells (F) in vemurafenib resistant WM793 cells treated with and without vemurafenib and/or PF562711 ( n = 3). Scale bars = 10 μm. ** P < 0.01; *** P < 0.001.

Journal: Cancer Biology & Medicine

Article Title: PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas

doi: 10.20892/j.issn.2095-3941.2020.0294

Figure Lengend Snippet: The PYK2 inhibitor, PF562711, abrogates vemurafenib-induced invadopodia formation and cell invasion. (A–C) Western blot analysis of p-PYK2 levels (A), representative images of fluorescence staining of actin (B), and quantification of invadopodia (C) in WM793 cells treated with dimethyl sulfoxide or PF562711 ( n = 3). (D) Quantification of invadopodia in WM793 cells treated with and without vemurafenib and/or PF562711 ( n = 3). (E, F) Quantification of invadopodia (E) and invaded cells (F) in vemurafenib resistant WM793 cells treated with and without vemurafenib and/or PF562711 ( n = 3). Scale bars = 10 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: TMAs were immunohistochemically stained with mouse monoclonal anti-human p-PYK2-Y402 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Fluorescence, Staining

STIM1 mediates vemurafenib-induced PYK2 activation and invadopodia formation. (A) Western blot analysis of p-PYK2 and STIM1 expressions in STIM1 knockout (KO) and control WM793 cells treated with and without vemurafenib. (B) Quantification of invadopodia in STIM1 KO and control WM793 cells treated with and without vemurafenib ( n = 3). (C) Quantification of STIM1 mRNA levels in vemurafenib-resistant and naïve WM793 treated with and without vemurafenib. (D) Effects of thapsigargin on Ca 2+ influx in vemurafenib resistant and naïve WM793 cells. The results are representative of 3 replicates. (E) Western blot analyses of p-Pyk2 and STIM1 levels in control WM793 and vemurafenib-resistant WM793 cells treated with and without EGTA or W7. (F) Quantification of invadopodia in control WM793 and vemurafenib-resistant WM793 cells treated with and without EGTA or W7. *** P < 0.001.

Journal: Cancer Biology & Medicine

Article Title: PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas

doi: 10.20892/j.issn.2095-3941.2020.0294

Figure Lengend Snippet: STIM1 mediates vemurafenib-induced PYK2 activation and invadopodia formation. (A) Western blot analysis of p-PYK2 and STIM1 expressions in STIM1 knockout (KO) and control WM793 cells treated with and without vemurafenib. (B) Quantification of invadopodia in STIM1 KO and control WM793 cells treated with and without vemurafenib ( n = 3). (C) Quantification of STIM1 mRNA levels in vemurafenib-resistant and naïve WM793 treated with and without vemurafenib. (D) Effects of thapsigargin on Ca 2+ influx in vemurafenib resistant and naïve WM793 cells. The results are representative of 3 replicates. (E) Western blot analyses of p-Pyk2 and STIM1 levels in control WM793 and vemurafenib-resistant WM793 cells treated with and without EGTA or W7. (F) Quantification of invadopodia in control WM793 and vemurafenib-resistant WM793 cells treated with and without EGTA or W7. *** P < 0.001.

Article Snippet: TMAs were immunohistochemically stained with mouse monoclonal anti-human p-PYK2-Y402 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot, Knock-Out, Control

Expression of PYK2-Y402 in melanoma tissues and survival analysis of melanoma patients. (A, B) Representative images of p-PYK2 immunohistochemical staining in lymph node metastasis (A) and distant metastasis (B) of melanomas. (C) Representative images of p-PYK2 immunohistochemical staining of Clark grade II, III, IV, and V melanomas. (D) Comparison analyses of p-PYK2 expression levels in melanomas with different Clark grades. (E) The curves of overall survival according to p-PYK2 expressions in patients with melanomas. (F) The curves of progression-free survival according to p-PYK2 expressions in patients with melanomas. * P < 0.05; ** P < 0.01.

Journal: Cancer Biology & Medicine

Article Title: PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas

doi: 10.20892/j.issn.2095-3941.2020.0294

Figure Lengend Snippet: Expression of PYK2-Y402 in melanoma tissues and survival analysis of melanoma patients. (A, B) Representative images of p-PYK2 immunohistochemical staining in lymph node metastasis (A) and distant metastasis (B) of melanomas. (C) Representative images of p-PYK2 immunohistochemical staining of Clark grade II, III, IV, and V melanomas. (D) Comparison analyses of p-PYK2 expression levels in melanomas with different Clark grades. (E) The curves of overall survival according to p-PYK2 expressions in patients with melanomas. (F) The curves of progression-free survival according to p-PYK2 expressions in patients with melanomas. * P < 0.05; ** P < 0.01.

Article Snippet: TMAs were immunohistochemically stained with mouse monoclonal anti-human p-PYK2-Y402 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison

(A) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.1% DMSO; black) and PHE either alone (red) or with the PKC inhibitors chelerythrine (Chel; 10 µM; bright brown) and bisindolylmaleimide I (Bis I; 100 nM; dark brown), the Pyk2 inhibitor PF-719 (1 µM; green), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). (B) The increase in NPo by PHE was blocked by all inhibitors. F 6,108 =6.434. Control vs. PHE, P =0.0076; PHE vs. Chel+PHE, P =0.0001; PHE vs. Bis I+PHE, P =0.0076; PHE vs. PF- 719+PHE, P =0.0018; PHE vs. PP2+PHE, P =0.0003; PHE vs. SU6656+PHE, P =0.0022. (C) Ensemble averages during depolarization. (D) The increase in ensemble average peak currents by PHE was blocked by PKC inhibitors chelerythrine, bisinolylmaleimide I and Src inhibitor PP2. F 6,108 =4.839. Control vs. PHE, P =0.0242; PHE vs. Chel+PHE, P =0.0004; PHE vs. Bis I+PHE, P =0.0242; PHE vs. PF- 719+PHE, P =0.0723; PHE vs. PP2+PHE, P =0.0006; PHE vs. SU6656+PHE, P =0.0723. (B,D) Data are presented as means ± SEM. n represents the number of cells (* P ≤0.05, ** P ≤0.01, *** P ≤0.001; ANOVA with post-hoc Holm-Sidak’s multiple comparisons test).

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: (A) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.1% DMSO; black) and PHE either alone (red) or with the PKC inhibitors chelerythrine (Chel; 10 µM; bright brown) and bisindolylmaleimide I (Bis I; 100 nM; dark brown), the Pyk2 inhibitor PF-719 (1 µM; green), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). (B) The increase in NPo by PHE was blocked by all inhibitors. F 6,108 =6.434. Control vs. PHE, P =0.0076; PHE vs. Chel+PHE, P =0.0001; PHE vs. Bis I+PHE, P =0.0076; PHE vs. PF- 719+PHE, P =0.0018; PHE vs. PP2+PHE, P =0.0003; PHE vs. SU6656+PHE, P =0.0022. (C) Ensemble averages during depolarization. (D) The increase in ensemble average peak currents by PHE was blocked by PKC inhibitors chelerythrine, bisinolylmaleimide I and Src inhibitor PP2. F 6,108 =4.839. Control vs. PHE, P =0.0242; PHE vs. Chel+PHE, P =0.0004; PHE vs. Bis I+PHE, P =0.0242; PHE vs. PF- 719+PHE, P =0.0723; PHE vs. PP2+PHE, P =0.0006; PHE vs. SU6656+PHE, P =0.0723. (B,D) Data are presented as means ± SEM. n represents the number of cells (* P ≤0.05, ** P ≤0.01, *** P ≤0.001; ANOVA with post-hoc Holm-Sidak’s multiple comparisons test).

Article Snippet: For production of HIV viral particles targeting Src and Pyk2, cells were transfected at a ratio of 5:2:2:2 of parental vector (e.g. pGFP-Pyk2-shC-Lenti): pCI-VSVG: pMDL g/p RRE: pRSV-REV) according to manufacturer’s guidelines (OriGene).

Techniques:

(A) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.06% DMSO; black) and 2 µM PMA either alone (red) or with the Pyk2 inhibitors PF-719 (1 µM; green) and PF-431396 (3 µM; orange), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). (B) The increase in NPo by PMA was blocked by all inhibitors. F 5,130 =6.530. DMSO vs. PMA, P =0.0003; PMA vs. PF-719+PMA, P =0.0372, PMA vs. PF-431396+PMA, P =0.0009; PMA vs. PP2+PMA, P =0.0003; PMA vs. SU6656+PMA, P =0.0005. (C) Ensemble averages during depolarization. (D) The increase in ensemble average peak currents by PMA was blocked by all inhibitors. F 5,130 =5.665. DMSO vs. PMA, P =0.0003; PMA vs. PF-719+PMA, P =0.0303, PMA vs. PF- 431396+PMA, P =0.0051; PMA vs. PP2+PMA, P =0.0003; PMA vs. SU6656+PMA, P =0.0051. (B,D) Data are presented as means ± SEM. n represents the number of cells (* P ≤0.05, ** P≤ 0.01, *** P ≤0.001; ANOVA with post-hoc Holm-Sidak’s multiple comparisons test).

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: (A) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.06% DMSO; black) and 2 µM PMA either alone (red) or with the Pyk2 inhibitors PF-719 (1 µM; green) and PF-431396 (3 µM; orange), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). (B) The increase in NPo by PMA was blocked by all inhibitors. F 5,130 =6.530. DMSO vs. PMA, P =0.0003; PMA vs. PF-719+PMA, P =0.0372, PMA vs. PF-431396+PMA, P =0.0009; PMA vs. PP2+PMA, P =0.0003; PMA vs. SU6656+PMA, P =0.0005. (C) Ensemble averages during depolarization. (D) The increase in ensemble average peak currents by PMA was blocked by all inhibitors. F 5,130 =5.665. DMSO vs. PMA, P =0.0003; PMA vs. PF-719+PMA, P =0.0303, PMA vs. PF- 431396+PMA, P =0.0051; PMA vs. PP2+PMA, P =0.0003; PMA vs. SU6656+PMA, P =0.0051. (B,D) Data are presented as means ± SEM. n represents the number of cells (* P ≤0.05, ** P≤ 0.01, *** P ≤0.001; ANOVA with post-hoc Holm-Sidak’s multiple comparisons test).

Techniques:

(A,B) Co-immunoprecipitation of Pyk2 and Src with Ca V 1.2 from brain (A) and heart (B). Triton X-100 extracts were cleared from non-soluble material by ultracentrifugation before immunoprecipitation (IP) with antibodies against α 1 1.2, Pyk2 itself, or non-immune control antibodies (IgG) and immunoblotting (IB) with anti-Pyk2 and anti-Src. Brain lysate (A, Input; 20 μl) and Pyk2 immunoprecipitates (B) served as positive control for detection of Pyk2 and Src by IB. Lanes for IgG control and α 1 1.2 IP in B are from the same IB and exposure but rearranged to eliminate non-relevant lanes. Pyk2 IP in B is from the same IB but is shown as a shorter exposure than the IgG and α 1 1.2 IP lanes. Comparable results were obtained in 4 independent experiments. (C) Schematic diagram of the intracellular α 1 1.2 fragments used in the pulldown assay (Table S1). (D) Pulldown assay of Pyk2 binding to α 1 1.2 fragments. GST fusion proteins of the N-terminus, the loops between domains I and II, II and III, III and IV, the whole C-terminus, and three different overlapping fragments covering the C-terminus were expressed in E. coli , immobilized on glutathione Sepharose, washed and incubated with purified His-tagged Pyk2. Comparable amounts of fusion proteins were present (data not shown but see ( , , , )). Comparable results were obtained in 5 independent experiments.

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: (A,B) Co-immunoprecipitation of Pyk2 and Src with Ca V 1.2 from brain (A) and heart (B). Triton X-100 extracts were cleared from non-soluble material by ultracentrifugation before immunoprecipitation (IP) with antibodies against α 1 1.2, Pyk2 itself, or non-immune control antibodies (IgG) and immunoblotting (IB) with anti-Pyk2 and anti-Src. Brain lysate (A, Input; 20 μl) and Pyk2 immunoprecipitates (B) served as positive control for detection of Pyk2 and Src by IB. Lanes for IgG control and α 1 1.2 IP in B are from the same IB and exposure but rearranged to eliminate non-relevant lanes. Pyk2 IP in B is from the same IB but is shown as a shorter exposure than the IgG and α 1 1.2 IP lanes. Comparable results were obtained in 4 independent experiments. (C) Schematic diagram of the intracellular α 1 1.2 fragments used in the pulldown assay (Table S1). (D) Pulldown assay of Pyk2 binding to α 1 1.2 fragments. GST fusion proteins of the N-terminus, the loops between domains I and II, II and III, III and IV, the whole C-terminus, and three different overlapping fragments covering the C-terminus were expressed in E. coli , immobilized on glutathione Sepharose, washed and incubated with purified His-tagged Pyk2. Comparable amounts of fusion proteins were present (data not shown but see ( , , , )). Comparable results were obtained in 5 independent experiments.

Techniques: Immunoprecipitation, Western Blot, Positive Control, Binding Assay, Incubation, Purification

PC12 cells were pretreated with vehicle (0.02% DMSO), the Pyk2 inhibitor PF-431396 (3 μM), and the Src inhibitor PP2 (10 μM) or its inactive analogue PP3 (10 μM) for 5 min before application of bradykinin (Brad., 2 μM) or PMA (2 μM) for 10 min, extraction with 1% SDS at 65°C to ensure dissociation of all proteins, neutralization of SDS with excess of Triton X-100, and ultracentrifugation. Supernatants were analysed by direct IB with the indicated Pyk2 and Src antibodies. Some samples underwent IP with the anti-phosphotyrosine antibody 4G10 before IB with anti-Pyk2 antibody (top panel in A and quantification in B). IgG indicates control IP with non-immune mouse IgG. (A) Upper panel: total pY levels of Pyk2 determined by IP with 4G10 and IB with anti-Pyk2. Middle panel: pY402 levels of Pyk2 detected with anti-pY402 in corresponding lysates. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (B) Ratios of total pY of Pyk2 after 4G10 IP to total Pyk2 in lysates, normalized to control. F 5,63 =12.73. DMSO vs. Brad., P =0.012; DMSO vs. PMA, P <0.0001; Brad. vs PF-431396+Brad., P <0.0001; PMA vs. PF-431396+PMA, P =0.0001. (C) Ratios of pY402 to total Pyk2 signals in lysates, normalized to control. F 5,35 =10.94. DMSO vs. Brad., P =0.039; DMSO vs. PMA, P =0.0052; Brad. vs PF-431396+Brad., P =0.0005; PMA vs. PF-431396+PMA, P <0.0001. (D) Upper panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (E) Ratios of pY579 to total Pyk2 signals in lysates, normalized to control. F 5,36 =10.18. DMSO vs. Brad., P =0.0072; DMSO vs. PMA, P =0.021; Brad. vs PF-431396+Brad., P =0.0008; PMA vs. PF-431396+PMA, P =0.0011. (F,H) Upper panels: pY416 levels of Src detected with anti-pY416. Lower panels: Levels of total Src detected with anti-Src in same lysates. (G,I) Ratios of pY416 to total Src signals in lysates, normalized to control. (G) F 5,72 =4.464. DMSO vs. Brad., P =0.0167; DMSO vs. PMA, P =0.0226. (I) F 5,65 =11.06. DMSO vs. PMA, P =0.001; PMA vs. PP2+PMA, P <0.0001; DMSO vs. PP3+PMA, P =0.0042; PP3 vs. PP3+PMA, P =0.0086. (J) Upper panel: pY402 levels of Pyk2 detected with anti-pY402. Middle panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (K,L) Ratios of pY402 and pY579 to total Pyk2 signals in lysates, normalized to control. (K) F 5,42 =35.85. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PP3 vs. PP3+PMA, P =0.0001; DMSO vs. PP3+PMA, P =0.0068; DMSO vs. PP2+PMA, P =0.0001; DMSO vs. PP2, P <0.0001. (L) F 5,68 =13.40. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PP3 vs. PP3+PMA, P =0.0362; DMSO vs. PP3+PMA, P =0.0202. (B,C,E,G,I,K,L) Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis was by ANOVA with post-hoc Bonferroni’s multiple comparisons test; * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. Bradykinin and PMA induced phosphorylation of Pyk2 on Y402 and Y579 and of Src on Y416, all of which were blocked by PF-431396 and PP2 but not the inactive PP3.

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: PC12 cells were pretreated with vehicle (0.02% DMSO), the Pyk2 inhibitor PF-431396 (3 μM), and the Src inhibitor PP2 (10 μM) or its inactive analogue PP3 (10 μM) for 5 min before application of bradykinin (Brad., 2 μM) or PMA (2 μM) for 10 min, extraction with 1% SDS at 65°C to ensure dissociation of all proteins, neutralization of SDS with excess of Triton X-100, and ultracentrifugation. Supernatants were analysed by direct IB with the indicated Pyk2 and Src antibodies. Some samples underwent IP with the anti-phosphotyrosine antibody 4G10 before IB with anti-Pyk2 antibody (top panel in A and quantification in B). IgG indicates control IP with non-immune mouse IgG. (A) Upper panel: total pY levels of Pyk2 determined by IP with 4G10 and IB with anti-Pyk2. Middle panel: pY402 levels of Pyk2 detected with anti-pY402 in corresponding lysates. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (B) Ratios of total pY of Pyk2 after 4G10 IP to total Pyk2 in lysates, normalized to control. F 5,63 =12.73. DMSO vs. Brad., P =0.012; DMSO vs. PMA, P <0.0001; Brad. vs PF-431396+Brad., P <0.0001; PMA vs. PF-431396+PMA, P =0.0001. (C) Ratios of pY402 to total Pyk2 signals in lysates, normalized to control. F 5,35 =10.94. DMSO vs. Brad., P =0.039; DMSO vs. PMA, P =0.0052; Brad. vs PF-431396+Brad., P =0.0005; PMA vs. PF-431396+PMA, P <0.0001. (D) Upper panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (E) Ratios of pY579 to total Pyk2 signals in lysates, normalized to control. F 5,36 =10.18. DMSO vs. Brad., P =0.0072; DMSO vs. PMA, P =0.021; Brad. vs PF-431396+Brad., P =0.0008; PMA vs. PF-431396+PMA, P =0.0011. (F,H) Upper panels: pY416 levels of Src detected with anti-pY416. Lower panels: Levels of total Src detected with anti-Src in same lysates. (G,I) Ratios of pY416 to total Src signals in lysates, normalized to control. (G) F 5,72 =4.464. DMSO vs. Brad., P =0.0167; DMSO vs. PMA, P =0.0226. (I) F 5,65 =11.06. DMSO vs. PMA, P =0.001; PMA vs. PP2+PMA, P <0.0001; DMSO vs. PP3+PMA, P =0.0042; PP3 vs. PP3+PMA, P =0.0086. (J) Upper panel: pY402 levels of Pyk2 detected with anti-pY402. Middle panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (K,L) Ratios of pY402 and pY579 to total Pyk2 signals in lysates, normalized to control. (K) F 5,42 =35.85. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PP3 vs. PP3+PMA, P =0.0001; DMSO vs. PP3+PMA, P =0.0068; DMSO vs. PP2+PMA, P =0.0001; DMSO vs. PP2, P <0.0001. (L) F 5,68 =13.40. DMSO vs. PMA, P <0.0001; PMA vs. PP2+PMA, P <0.0001; PP3 vs. PP3+PMA, P =0.0362; DMSO vs. PP3+PMA, P =0.0202. (B,C,E,G,I,K,L) Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis was by ANOVA with post-hoc Bonferroni’s multiple comparisons test; * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. Bradykinin and PMA induced phosphorylation of Pyk2 on Y402 and Y579 and of Src on Y416, all of which were blocked by PF-431396 and PP2 but not the inactive PP3.

Techniques: Neutralization

(A) Lysates from HEK293T/17 cells transfected with vectors encoding rat Pyk2 ( rPyk2-GFP) and either the Pyk2-targeting FIV lentivirus-derived, pVETL-Sh1-GFP (pFV-Pyk2-Sh1) or control (empty) pVETL-GFP (pFV-GFP) expression vectors, were immunoblotted (IB) with indicated antibodies. (B,C) IB analysis of indicated proteins in PC12 cultures incubated with viral particles containing pFV-Sh1-GFP (Sh1) FIV-based expression vector used in A or medium vehicle alone for 72h prior to treatment with either PMA (B), bradykinin (Brad.; C) or vehicle alone (-; B,C). Upper blots in B&C show anti-α 1 1.2 IBs of 4G10-anti-phosphotyrosine (pY) IP while middle and lower blots show direct IBs of indicated protein levels in input lysates. (D) Statistical analysis of the relative pY α 1 1.2 levels. F 5,41 =8.276. NT vs. PMA, P =0.0031; NT vs. Brad., P =0.0017; PMA vs. Sh1+PMA, P =0.001; Brad vs. Sh1+Brad, P =0.0433. (E,F) Direct IB analysis of indicated proteins in lysates of PC12 cultures transduced with HIV vector-derived lentiviral particles (e.g., pGFP-Pyk2-ShB-Lenti) containing expression cassettes for GFP and either the Pyk2-targeting (denoted pHV-Pyk-ShB and -ShC), Src-targeting (denoted pHV-Src-ShC and -ShD) or scrambled hairpin control (Cont.) shRNAs. In some cases (right blot) cultures were treated with PMA (+) or vehicle alone (-) before harvesting for IB. (G,H) IB analysis of indicated proteins from PC12 cultures infected with lentiviral particles containing HIV-GFP expression vectors as in E&F prior to treatment with either PMA (+) or vehicle (-). Upper panels show anti -α 1 1.2 IBs of 4G10-anti-pY IP while lower blots show direct IBs of input lysates with indicated antibodies. (I) Statistical analysis of relative α 1 1.2 pY levels. F 11,129 =6.180. NT vs. PMA, P <0.0001; PMA vs. Pyk2-ShB, P =0.0005; PMA vs. Pyk2-ShB+PMA, P =0.0029; PMA vs. Pyk2-ShC, P <0.0001; PMA vs. Pyk2-ShC+PMA, P =0.0002; PMA vs. Cont.-Sh, P =0.0019; PMA vs. Cont.-Sh+PMA, P >0.9999; PMA vs. Src-ShB, P =0.0007; PMA vs. Src-ShB+PMA, P <0.0001; PMA vs. Src-ShC, P <0.0001; PMA vs. Src-ShC+PMA, P< 0.0001. The bar graphs in (D) and (I) show ratios of quantified anti-α 1 1.2 IB signals in 4G10 IPs relative to α 1 1.2 IB signals in total lysates, normalized to not treated (NT) control. Comparisons are made between samples treated with PMA (or bradykinin in D) and each of the other indicated conditions. Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis by ANOVA with post-hoc Bonferroni’s multiple comparisons test (ns= not significant vs. PMA , *P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001).

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: (A) Lysates from HEK293T/17 cells transfected with vectors encoding rat Pyk2 ( rPyk2-GFP) and either the Pyk2-targeting FIV lentivirus-derived, pVETL-Sh1-GFP (pFV-Pyk2-Sh1) or control (empty) pVETL-GFP (pFV-GFP) expression vectors, were immunoblotted (IB) with indicated antibodies. (B,C) IB analysis of indicated proteins in PC12 cultures incubated with viral particles containing pFV-Sh1-GFP (Sh1) FIV-based expression vector used in A or medium vehicle alone for 72h prior to treatment with either PMA (B), bradykinin (Brad.; C) or vehicle alone (-; B,C). Upper blots in B&C show anti-α 1 1.2 IBs of 4G10-anti-phosphotyrosine (pY) IP while middle and lower blots show direct IBs of indicated protein levels in input lysates. (D) Statistical analysis of the relative pY α 1 1.2 levels. F 5,41 =8.276. NT vs. PMA, P =0.0031; NT vs. Brad., P =0.0017; PMA vs. Sh1+PMA, P =0.001; Brad vs. Sh1+Brad, P =0.0433. (E,F) Direct IB analysis of indicated proteins in lysates of PC12 cultures transduced with HIV vector-derived lentiviral particles (e.g., pGFP-Pyk2-ShB-Lenti) containing expression cassettes for GFP and either the Pyk2-targeting (denoted pHV-Pyk-ShB and -ShC), Src-targeting (denoted pHV-Src-ShC and -ShD) or scrambled hairpin control (Cont.) shRNAs. In some cases (right blot) cultures were treated with PMA (+) or vehicle alone (-) before harvesting for IB. (G,H) IB analysis of indicated proteins from PC12 cultures infected with lentiviral particles containing HIV-GFP expression vectors as in E&F prior to treatment with either PMA (+) or vehicle (-). Upper panels show anti -α 1 1.2 IBs of 4G10-anti-pY IP while lower blots show direct IBs of input lysates with indicated antibodies. (I) Statistical analysis of relative α 1 1.2 pY levels. F 11,129 =6.180. NT vs. PMA, P <0.0001; PMA vs. Pyk2-ShB, P =0.0005; PMA vs. Pyk2-ShB+PMA, P =0.0029; PMA vs. Pyk2-ShC, P <0.0001; PMA vs. Pyk2-ShC+PMA, P =0.0002; PMA vs. Cont.-Sh, P =0.0019; PMA vs. Cont.-Sh+PMA, P >0.9999; PMA vs. Src-ShB, P =0.0007; PMA vs. Src-ShB+PMA, P <0.0001; PMA vs. Src-ShC, P <0.0001; PMA vs. Src-ShC+PMA, P< 0.0001. The bar graphs in (D) and (I) show ratios of quantified anti-α 1 1.2 IB signals in 4G10 IPs relative to α 1 1.2 IB signals in total lysates, normalized to not treated (NT) control. Comparisons are made between samples treated with PMA (or bradykinin in D) and each of the other indicated conditions. Data are presented as mean ± SEM. Number ( n ) of independent experiments for each condition are indicated inside bars. Statistical analysis by ANOVA with post-hoc Bonferroni’s multiple comparisons test (ns= not significant vs. PMA , *P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001).

Techniques: Transfection, Derivative Assay, Expressing, Incubation, Plasmid Preparation, Transduction, Infection

LTP LTCC was induced by four 200 Hz tetani, each 0.5 s long, in the CA1 Schaffer collateral projections to CA1 in acute hippocampal slices from 13-20 weeks old mice. (A) LTP LTCC required PHE (10 μM) and was prevented by the LTCC blocker nimodipine (10 μM; NIMO) and the α 1 AR antagonist prazosin (1 μM; PRAZ). F 3,36 =9.937. Control vs. PHE, P =0.012; PHE vs. PHE/PRAZ, P =0.0001; PHE vs. PHE/NIMO, P =0.0003. (B) PHE-mediated LTP is blocked by inhibitors of Pyk2 (1 μM PF-719) and Src (10 μM PP2). F 2,30 =13.90. PHE vs. PHE/PF-719, P =0.0002; PHE vs. PHE/PP2, P =0.0003. Dot plots on the right show potentiation of fEPSPs determined as the averages of all responses between 45 and 50 min after high frequency stimulation (HFS) as % of averages of all responses in the 5 min preceding HFS. Bars and whiskers represent means ± SEM (* P ≤0.05, *** P ≤0.001; one-way ANOVA with the Bonferroni correction). The number of slices and mice used are indicated.

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet: LTP LTCC was induced by four 200 Hz tetani, each 0.5 s long, in the CA1 Schaffer collateral projections to CA1 in acute hippocampal slices from 13-20 weeks old mice. (A) LTP LTCC required PHE (10 μM) and was prevented by the LTCC blocker nimodipine (10 μM; NIMO) and the α 1 AR antagonist prazosin (1 μM; PRAZ). F 3,36 =9.937. Control vs. PHE, P =0.012; PHE vs. PHE/PRAZ, P =0.0001; PHE vs. PHE/NIMO, P =0.0003. (B) PHE-mediated LTP is blocked by inhibitors of Pyk2 (1 μM PF-719) and Src (10 μM PP2). F 2,30 =13.90. PHE vs. PHE/PF-719, P =0.0002; PHE vs. PHE/PP2, P =0.0003. Dot plots on the right show potentiation of fEPSPs determined as the averages of all responses between 45 and 50 min after high frequency stimulation (HFS) as % of averages of all responses in the 5 min preceding HFS. Bars and whiskers represent means ± SEM (* P ≤0.05, *** P ≤0.001; one-way ANOVA with the Bonferroni correction). The number of slices and mice used are indicated.

Techniques:

Journal: bioRxiv

Article Title: α 1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca 2+ channel Ca v 1.2 activity and long-term potentiation in rodents

doi: 10.1101/2022.07.01.498400

Figure Lengend Snippet:

Techniques: shRNA

Figure 4. Effect of Orai1 suppression on C6 glioma cell adhesion and Pyk2 phosphorylation. shScramble or shOrai1 was introduced into C6 glioma cells, or cells were treated with SKF96365 (20 µM). (a) The relative cell adhesion was detected. (b) Cell morphology was visualized by immunofluorescence assay and the proportions of cells with large focal adhesions were calculated.(c) The protein levels of total Pyk2 and phosphorylated Pyk2 (p-Pyk2) were measured by western blot analysis. Data are shown as means ± SDs from thrice independent experiments. * P < 0.05 or ** P < 0.01 vs. the shScramble group.

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Suppression of store-operated Ca 2+ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion.

doi: 10.1080/15384101.2020.1843814

Figure Lengend Snippet: Figure 4. Effect of Orai1 suppression on C6 glioma cell adhesion and Pyk2 phosphorylation. shScramble or shOrai1 was introduced into C6 glioma cells, or cells were treated with SKF96365 (20 µM). (a) The relative cell adhesion was detected. (b) Cell morphology was visualized by immunofluorescence assay and the proportions of cells with large focal adhesions were calculated.(c) The protein levels of total Pyk2 and phosphorylated Pyk2 (p-Pyk2) were measured by western blot analysis. Data are shown as means ± SDs from thrice independent experiments. * P < 0.05 or ** P < 0.01 vs. the shScramble group.

Article Snippet: The PVDF membranes were then incubated with primary antibodies as follows: rabbit monoclonal anti-Orai1 (ab11196, Abcam, Cambridge, MA, USA, 1:1000), mouse monoclonal anti-proline-rich tyrosine kinase 2 (Pyk2, sc-393,181, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500), mouse monoclonal antiphosphorylated Pyk2 (p-Pyk2, MAB6210-SP, R&D Systems, Minneapolis, MN, USA, 0.5 μg/mL), rabbit monoclonal anti-p21(ab109520, Abcam, 1:2000), rabbit monoclonal anti-Cyclin D1 (ab40754, Abcam, 1:2000), rabbit monoclonal anti-CDK4 (ab108357, Abcam, 1:2000), rabbit monoclonal anti-E-cadherin (ab133597, Abcam, 1:2000), rabbit polyclonal antiN-cadherin (ab76057, Abcam, 1:1000), rabbit polyclonal anti-Vimentin (ab137321, Abcam, 1:2000), rabbit polyclonal anti-p-AKT (ab38449, Abcam, 1:1000), rabbit polyclonal anti-p-4EBP1 (ab47365, Abcam, 1:1000), rabbit polyclonal anti-NFAT (ab3447, Abcam, 1:1000), rabbit polyclonal anti-NF-κB (ab16502, Abcam, 0.5 μg/mL), rabbit polyclonal antip-S6K (ab59208, Abcam, 1:1000), and rabbit monoclonal anti-GAPDH (Abcam).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot

SKAP2 is required for optimal C. glabrata-stimulated phosphorylation of Pyk2 but not Syk kinases WT and Skap2−/− neutrophils were (A–C and G–I) plated onto immobilized TDB, furfurman (Fur), or IgG immune complexes (IC), or (D–F and J–L) stimulated with MOI 2 of C. albicans and C. glabrata for 15 min at 37°C. Lysates were analyzed by Western blot for pSyk (Y352), pPyk2 (Y402), and RhoGDI, then stripped, and re-probed for respective total proteins. Quantification is shown in . Data were compiled from 3-5 independent experiments. (A, D, G, and J) Representative blot shown with arrows to indicate bands used for analysis.(B, E, H, and K) Fold change from each experiment with respect to unstimulated, symbols represent the value from each experiment, solid symbols indicating values of blot shown, bars indicate mean. Statistics represent mean ± SEM. (C, F, I, and L) The Skap2−/− values were divided by WT values in each respective experiment. The average WT level of CLR-stimulated phosphorylation was set to 1 as indicated by the dotted line. Data were compiled from n = 3–5 independent experiments with data presented as mean ± SEM. Significance was assessed using (B, E, and H) two-way ANOVA with Tukey's post-test or (K) one-way ANOVA within each group, or two-way ANOVA between WT and KO groups with Sidak’s post-test. See also <xref ref-type=

Figure S4 and . " width="100%" height="100%">

Journal: iScience

Article Title: Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

doi: 10.1016/j.isci.2021.102871

Figure Lengend Snippet: SKAP2 is required for optimal C. glabrata-stimulated phosphorylation of Pyk2 but not Syk kinases WT and Skap2−/− neutrophils were (A–C and G–I) plated onto immobilized TDB, furfurman (Fur), or IgG immune complexes (IC), or (D–F and J–L) stimulated with MOI 2 of C. albicans and C. glabrata for 15 min at 37°C. Lysates were analyzed by Western blot for pSyk (Y352), pPyk2 (Y402), and RhoGDI, then stripped, and re-probed for respective total proteins. Quantification is shown in . Data were compiled from 3-5 independent experiments. (A, D, G, and J) Representative blot shown with arrows to indicate bands used for analysis.(B, E, H, and K) Fold change from each experiment with respect to unstimulated, symbols represent the value from each experiment, solid symbols indicating values of blot shown, bars indicate mean. Statistics represent mean ± SEM. (C, F, I, and L) The Skap2−/− values were divided by WT values in each respective experiment. The average WT level of CLR-stimulated phosphorylation was set to 1 as indicated by the dotted line. Data were compiled from n = 3–5 independent experiments with data presented as mean ± SEM. Significance was assessed using (B, E, and H) two-way ANOVA with Tukey's post-test or (K) one-way ANOVA within each group, or two-way ANOVA between WT and KO groups with Sidak’s post-test. See also Figure S4 and .

Article Snippet: Rabbit anti-human/mouse polyclonal Pyk2 , Cell Signaling Technology , Cell Signaling Technology Cat# 3292, RRID: AB_2174097.

Techniques: Phospho-proteomics, Western Blot

SKAP2 functions prior to or after Syk activation after infection with different pathogens The binding of K. pneumoniae to surface receptors on neutrophils leads to the SKAP2-mediated phosphorylation of Syk and/or Pyk2 and requires the production of extracellular ROS. The binding of C. glabrata and C. albicans does not require SKAP2 for Syk phosphorylation; however, Pyk2 phosphorylation was dependent on SKAP2. Binding by the CLR agonist, furfurman and TBD induces Syk and Pyk phosphorylation independently of SKAP2. Likewise, SKAP2 contributes to extracellular ROS production and cell adhesion following the stimulation of integrins and fMLP ( <xref ref-type=

Nguyen et al., 2020 ; Boras et al., 2017 ; Shaban et al., 2020 ). " width="100%" height="100%">

Journal: iScience

Article Title: Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

doi: 10.1016/j.isci.2021.102871

Figure Lengend Snippet: SKAP2 functions prior to or after Syk activation after infection with different pathogens The binding of K. pneumoniae to surface receptors on neutrophils leads to the SKAP2-mediated phosphorylation of Syk and/or Pyk2 and requires the production of extracellular ROS. The binding of C. glabrata and C. albicans does not require SKAP2 for Syk phosphorylation; however, Pyk2 phosphorylation was dependent on SKAP2. Binding by the CLR agonist, furfurman and TBD induces Syk and Pyk phosphorylation independently of SKAP2. Likewise, SKAP2 contributes to extracellular ROS production and cell adhesion following the stimulation of integrins and fMLP ( Nguyen et al., 2020 ; Boras et al., 2017 ; Shaban et al., 2020 ).

Article Snippet: Rabbit anti-human/mouse polyclonal Pyk2 , Cell Signaling Technology , Cell Signaling Technology Cat# 3292, RRID: AB_2174097.

Techniques: Activation Assay, Infection, Binding Assay, Phospho-proteomics

Journal: iScience

Article Title: Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

doi: 10.1016/j.isci.2021.102871

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human/mouse polyclonal Pyk2 , Cell Signaling Technology , Cell Signaling Technology Cat# 3292, RRID: AB_2174097.

Techniques: Blocking Assay, Virus, Recombinant, Clinical Proteomics, Prestoblue Assay, Software

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