44-618G

antibody from Invitrogen Antibodies

Targeting: PTK2B CADTK, CAKB, FAK2, PTK, PYK2, RAFTK

Provider product page for 44-618G

Western blot

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [27]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [2]
Flow cytometry [1]
Other assay [22]

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Product number: 44-618G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-PYK2 (Tyr402) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This product contains enough material for 10 mini-blots. Antibody has been negatively preadsorbed using a nonphosphopeptide then purified by epitope-specific affinity chromatography. The antibody has been used in western blotting and immunohistochemistry. Other applications may work but have not been tested. Positive controls used were CEF cell extracts +/- Pyk2 transfection or A431 whole cell lysate.
Reactivity: Human, Mouse, Rat, Chicken/Avian
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

PTK2B protein structure - 44-618G shown in red.

Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Proline-rich tyrosine kinase 2 phosphorylation induced by acute stretch is inhibited by PF-4594755 (PF). Rat portal veins were cultured with either DMSO (0.08%) or PF (1 mu mol/L) overnight (A-D). Following overnight equilibration, portal vein strips were stretched by a hanging weight for 10 min and quickly frozen in liquid nitrogen. Phosphorylation of PYK2 (A), Akt (B), FAK (C), and ERK1/2 (D) was determined with phospho-specific antibodies and the signal intensity normalized to the signal intensity of the respective total protein ( n = 7-10). * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant, for comparison with unloaded strips.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Interactions between PF-4594755 and inhibitors of voltage/non-voltage-dependent Ca 2+ entry. Portal vein strips were incubated in organ culture with or without load for 3 days in the presence of [ 35 S]-methionine for the last 24 h (A-D). Effect of PF (0.5 mu mol/L), nifedipine (Nif, 1 mu mol/L), and 2-APB (30 mu mol/L) on PYK2 phosphorylation (A), and effect of PF on Akt (B) and ERK1/2 (C) phosphorylation was evaluated by western blot ( n = 5-6). Summarized data of SM22 alpha synthesis as measured by autoradiography (D; n = 6). Effects of the combined action of PF and nifedipine or 2-APB on PYK2 phosphorylation compared with that of PF alone, normalized to phosphorylation of untreated control as in panel A (E; n = 3-4). DNA synthesis as measured by [ 3 H]-thymidine incorporation in portal veins cultured unloaded/loaded for 3 days with or without PF (F; n = 6). PYK2, Akt and ERK1/2 phosphorylation of portal vein cultured unloaded for 24 h or 72 h as indicated. (F, n = 5). In each panel, all blots shown are from the same gels for phosphorylated or total protein, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001 for comparison with unloaded strip, # P < 0.05, ## P < 0.01 for comparison with untreated control (unloaded/loaded).

Supportive validation

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Experimental details: Figure 1 Hyperactivation of PYK2 in ADM and PanIN lesions. ( A ) Human PDAC tissue was stained with antibodies against PYK2 and p-PYK2 Y402 . Representative IHC staining is shown. Scale bars : 100 mum. ( B ) IHC staining of PYK2 and p-PYK2 Y402 in pancreas from 6-month-old Pdx-Cre mice and Pdx-Cre ; LSL-Kras G12D mice. Scale bars : 50 mum. ( C ) Immunoblotting analysis of PYK2 and p-PYK2 Y402 levels in pancreatic tissues from Pdx-Cre control mice and Pdx-Cre ; LSL-Kras G12D mice. Each lane represents a single mouse. ( D ) C57BL/6 mice were injected with cerulein (to induce pancreatitis) or PBS (control) for 2 consecutive days. The pancreatic tissues were collected 2 days after injection and prepared for immunoblotting analysis with indicated antibodies. ( E ) Six-week-old C57BL/6 mice were treated with PBS or cerulein for 2 consecutive days. The pancreas was harvested at the indicated time points after injection for H&E staining and IHC staining. Scale bars : 50 mum. ( F ) Co-immunofluorescence staining for p-PYK2 Y402 and CK19 in pancreatic sections from cerulein- and PBS-treated mice. Scale bars : 50 mum. For all panels, representative results from 3 independent experiments are shown. ( C and D ) The numbers under each blot represent band intensity normalized to alpha-tubulin and relative to expression of target proteins in Pdx-Cre mice or PBS-treated C57BL/6 mice.

Supportive validation

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Experimental details: Fig. 1. TRPM2 depletion significantly increases doxorubicin sensitivity and reduces Pyk2 and CREB phosphorylation and expression. A : two different SH-SY5Y clones in which TRPM2 was depleted with CRISPR (KO) or scrambled control cells (Scr) were studied. Cells were untreated or treated with 0.3 muM doxorubicin for 24 or 48 h. Cell proliferation was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Results are expressed as OD reading of plated cells (5 x 10 4 cells) normalized to time 0 for each group. Values are means +- SE for one representative experiment analyzed in triplicate. Four experiments were performed. *Significantly different at P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2. TRPM2 inhibition with TRPM2-S significantly increases doxorubicin sensitivity and reduces Pyk2 phosphorylation and Pyk2 and CREB expression. A : SH-SY5Y cells expressing empty vector (V), TRPM2-L (L) or TRPM2-S (S) were untreated or treated with 0.5 muM doxorubicin for 24 or 48 h. Cell proliferation was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Results are expressed as OD reading of plated cells (5 x 10 4 cells) normalized to control at time 0 for each group. Values are means +- SE for one representative experiment analyzed in triplicate of two performed. *Significant differences at P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4. Pyk2 regulates CREB expression and cell viability. A : time course. SH-SY5Y cells stably expressing V5-TRPM2-L were transiently transfected with shRNA targeting Pyk2, and samples for Western blotting removed from culture at 24-h intervals for 168 h. Western blotting was performed with antibodies to V5, pPyk2, Pyk2, pCREB, and CREB. Tubulin was probed to confirm equivalent loading. B : SH-SY5Y cells expressing V5-TRPM2-L were stably transfected with shRNA targeting Pyk2 or control scrambled shRNA. Western blotting was performed on lysates from untreated cells with antibodies to V5, pPyk2, Pyk2, pCREB, and total CREB and confirmed that down modulation of Pyk2 resulted in reduced pPyk2, pCREB, and CREB. A representative Western blot from one of three experiments is shown. C : expression of pPyk2, Pyk2, pCREB, CREB, and tubulin was normalized by comparison of expression in cells transfected with Pyk2 targeted shRNA to that in scrambled shRNA for each densitometry measurement in the three experiments in B . The Student's t -test was used for analysis of differences. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5. Pyk2 rescues viability of TRPM2-S expressing cells. SH-SY5Y cells expressing V5-TRPM2-L or TRPM2-S were transfected with vector, Y402F Pyk2, Pyk2, or wild-type Pyk2. Cells were then treated with 0.3 muM doxorubicin for 24 or 48 h. A : cell proliferation was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Results are expressed as relative viability calculated by OD reading of plated cells (5 x 10 4 cells) normalized to time 0 for each group. Values are means +- SE for one representative experiment of three done in six replicates. Results were analyzed with one-way ANOVA. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6. Depletion of TRPM2 reduces mitochondrial phosphorylation of Src and Pyk2 and mitochondrial expression of Pyk2, CREB, and MCU. A : TRPM2-depleted and scrambled SH-SY5Y cells were separated into cytosol and mitochondrial fractions and Src, Pyk2, and CREB phosphorylation and expression examined. In both whole cell lysates and mitochondria, phosphorylation of Src and Pyk2 was decreased after doxorubicin treatment of TRPM2-depleted cells. Mitochondrial Pyk2 was also decreased after doxorubicin application. Levels of CREB and the mitochondrial calcium uniporter MCU were reduced in the mitochondrial fraction of KO cells. Similar results were observed in three experiments and representative blots are shown. Densitometry measurements of mitochondrial protein for three experiments were standardized to results for each experiment's scrambled mitochondrial control at time 0 , and the means +- SE of phosphorylated or total Src, Pyk2, CREB, or MCU calculated from three experiments are shown. ** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 10. Reconstitution of TRPM2-depleted cells with wild-type TRPM2 but not the Ca 2+ -impermeable mutant E960D restored viability in TRPM2-depleted cells and phosphorylation and expression of Pyk2 and CREB. TRPM2-depleted SH-SY5Y cells (KO) were stably transfected with empty vector, V5-tagged wild-type TRPM2 or the V5-tagged Ca 2+ -impermeable TRPM2 mutant E960D. Scrambled control cells (Scr) were transfected with empty vector. Two different single cells clones from each group of transfected cells (Scr, KO) were untreated or treated with doxorubicin for 24 h and cell lysates prepared. A : reconstitution of cell viability with wild-type TRPM2 but not empty vector or the E960D pore mutant is shown. Viability was measured in three experiments with XTT. Measurements were standardized to results for untreated cells in each group, and the means +- SE of six replicates from one representative experiment are shown. * P