- 44-636G - Invitrogen Antibodies PTK2B antibody

Submitted references Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH.

Wilson AD, Richards MA, Curtis MK, Gunadasa-Rohling M, Monterisi S, Loonat AA, Miller JJ, Ball V, Lewis A, Tyler DJ, Moshnikova A, Andreev OA, Reshetnyak YK, Carr C, Swietach P
Cardiovascular research 2022 Nov 10;118(14):2946-2959

Microglial Cytokines Induce Invasiveness and Proliferation of Human Glioblastoma through Pyk2 and FAK Activation.

Nuñez RE, Del Valle MM, Ortiz K, Almodovar L, Kucheryavykh L
Cancers 2021 Dec 7;13(24)

Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY
PloS one 2015;10(6):e0131059

Collagen XV inhibits epithelial to mesenchymal transition in pancreatic adenocarcinoma cells.

Clementz AG, Mutolo MJ, Leir SH, Morris KJ, Kucybala K, Harris H, Harris A
PloS one 2013;8(8):e72250

Analyzing FAK and Pyk2 in early integrin signaling events.

Bernard-Trifilo JA, Lim ST, Hou S, Schlaepfer DD, Ilic D
Current protocols in cell biology 2006 Apr;Chapter 14:Unit 14.7

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-FAK2 / PYK2 (Tyr580) was done on 70% confluent log phase A549 cells treated with 50 ng of PDGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-FAK2 / PYK2 (Tyr580) Rabbit Polyclonal Antibody (Product # 44-636G) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing membranous localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-Pyk2 (pYpY579/580) showing staining in the cytoplasm and nucleus of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Pyk2 (pYpY579/580) polyclonal antibody (Product # 44-636G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-Pyk2 (pYpY579/580) showing staining in the cytoplasm and nucleus of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Pyk2 (pYpY579/580) polyclonal antibody (Product # 44-636G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-Pyk2 (pYpY579/580) showing staining in the cytoplasm and nucleus of paraffin-embedded human spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Pyk2 (pYpY579/580) polyclonal antibody (Product # 44-636G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-Pyk2 (pYpY579/580) showing staining in the cytoplasm and nucleus of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Pyk2 (pYpY579/580) polyclonal antibody (Product # 44-636G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of FAK2 / PYK2 [pTyr580] was done on PC-3 cells treated with Vanadate (100uM, 1 hour). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with FAK2 / PYK2 [pTyr580] Rabbit Polyclonal Antibody (44636G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of PYK2 [pY580] was done on PC-3 cells treated with Pervanadate (100uM, 1 hour) . Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with PYK2 [pY580] Rabbit Polyclonal Antibody (44636G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Collagen XV inhibits phosphorylation of Pyk2 and suppresses N-Cadherin. Vector-only (BxVC3, BxVC4, BxVC5, BxVC10) and COLXV-expressing clones (Bx15.5, Bx15.14, Bx15.23, Bx15.24) were grown on COLI coated plates (A, B,); plastic and COLI (C) or plastic alone (F) and lysed after 48 h. Western blots probed with antibodies specific for A) total FAK or pFAK; B) total Pyk2 or pPyk2; C, F) N-Cad; are shown. ss-tubulin provides an estimate of total protein. Expression of COLXV slightly enhances pFAK relative to FAK (though this is not statistically significant, p = 0.0913), but depresses pPyk2 relative to Pyk2 (* p = 0.0447) and suppresses N-Cadherin levels. Blots were quantified by integrating data acquired with the Lasso tool of Adobe Photoshop (C, D, E, F) and analyzed by students t test (E, *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 3 Factors released from microglia upregulate phosphorylation of Pyk2 in glioma cells. Western blot analysis of pPyk2 (Tyr 579/580) protein in glioma cell lines. The signal is detected at the area corresponding to molecular weight of 116 kDa. The graph shows the density of protein in MCM and AMCM treatments relative to control. Two bands in pPyk2 (Tyr 579/580) detection identify phosphorylation in one or both sites. Intensity of the chemiluminescence signal was corrected for minor changes in protein content after densitometry analysis of the India ink stained membrane. The India Ink stained membranes are provided in S1 Fig . The ""relative density"" axis for HS683 cells is shown in a higher grid scale compare to other cell lines due to higher relative up regulation of Pyk2 phosphorylation in this cell line. Results are presented as mean +- S.D. with significant differences from control (*) (p < 0.05). One-way ANOVA followed by the Tukey's multiple comparison test was used to determine significance between MCM or AMCM groups compared to control. 5 repeated experiments (N = 5) for each cell line were used for statistical analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 5 Local microglia ablation in tumor area reduces phosphorylation of Pyk2 in implanted GL261 glioma tumors in CD11b-HSVTK transgenic mice. (A) Western blot detection of pPyk2 (Tyr 579/580) protein in glioma cells extracted from C57BL/6 and HSVTK-CD11b mice brains after treatment with GCV. Glioma cells were purified using Percoll gradients. (B) The graph shows the quantification of corresponding levels of pPyk2(Tyr 579/580) detected by western blot. Mean +- S.E and significant difference from control (*) is shown (p < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 2 Pyk2 is mostly detected in glioma cells rather than in other cell types in mouse brain. Immunohistochemistry was performed on C57BL/6 mice brain sections containing the tumor area. GL261 glioma cells were implanted into the brains of C57BL/6 mice and grown for 16 days. Photographs show the tumor and surrounding healthy tissue. The dash line outlines the border of tumor. Anti-GFAP antibody was used to detect glioma cells and astrocytes (red, panel A), anti-Iba 1 antibody was used to detect microglial cells (green, panel B) and Pyk2 detection is presented in blue (panel C). The merged images of anti-GFAP and anti-Pyk2, of anti-Iba1 and anti-Pyk2, and of all antibodies together can be seen in merged image boxes D, E, F correspondingly. Insert panels d , e , and f represent enlarged images of astrocytes, microglia, and invading glioma cells. Solid arrows indicate glioma cells, frame arrows indicate astrocytes, and double headed arrows indicate microglia. Scale bar: 40 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Cytokines and chemokines released by microglia upregulate Pyk2 and FAK protein phosphorylation in human glioma cells. Representative western blots and quantitative results for total and phosphorylated Pyk2 (Y579/580) and FAK (Y925) are presented for the cell lines CL1 ( a - d ), CL2 ( e - h ), and CL3 ( i - l ). The degree of phosphorylation was calculated as the ratio of phosphorylated Pyk2 or FAK to the total loaded protein and normalized to the control for each kinase. Total protein-stained membranes are provided in Figure S5 . The values are shown as means +- SD for 3-6 experiments per group. * p < 0.05 vs. control; ** p < 0.05 vs. MCM. MCM, microglia-conditioned medium; Inh III, inhibitor III; Bur, burixafor; Toc, tocilizumab; Rep, reparixin; and Gef, gefitinib.

44-636G

Antibody data