recombinant pyk2 protein  (Creative BioMart)

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

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: Journal of cellular biochemistry

Article Title: Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

doi: 10.1002/jcb.25430

Figure Lengend Snippet: Fig. 1. Osteoblast (OB) differentiation is impaired in the absence of Pyk2. Two day calvarial osteoblasts were generated from WT and Pyk2-KO mice. (A) Osteoblasts were cultured under osteogenic conditions containing ascorbic acid and b-glycerolphosphate for 7 days and then stained for alkaline phosphatase expression or cultured for 21 days and stained with Alizarin-S red as a measure of osteoblast mineralization. (B) QPCR analysis was used to determine mRNA expression of osteoblastic genes; ALP, type I collagen, and osteocalcin. GAPDH was used as the control to normalize the amount of the mRNA transcript under investigation. The mean of duplicate samples is shown for each transcript standard error of the mean (SEM). Experiments were performed a minimum of three times and representative data are shown. The asterisks () indicate statistical significance (P < 0.05) relative to control WT osteoblasts set at RQ 1.0.

Article Snippet: Recombinant adenoviruses expressing WT Pyk2 or its mutants were prepared by recombination of the above plasmids using the Adenovator Vector System (Qbiogene) according to the manufacturer0s instructions.

Techniques: Generated, Cell Culture, Staining, Expressing, Control

Journal: Journal of cellular biochemistry

Article Title: Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

doi: 10.1002/jcb.25430

Figure Lengend Snippet: Fig. 2. Pyk2-KO osteoblast (OB) differentiation is inhibited by Pyk2Y402F and Pyk2K457A, but not Pyk2WT. (A) WT osteoblasts were treated with 5 or 10 mm PAO for 1 h. Lysates were immunoprecipitated with anti-Pyk2 and blotted for phosphorylated tyrosine (p-Tyr) or using a phosho-specific p-Y402 antibody. Membranes were reblotted for total Pyk2 as a control. The Pyk2 antibody detects Pyk2WT, Pyk2Y402F, and Pyk2K457A. (B–C) Pyk2-KO osteoblasts were infected with adenoviruses expressing similar amounts of Pyk2WT

Article Snippet: Recombinant adenoviruses expressing WT Pyk2 or its mutants were prepared by recombination of the above plasmids using the Adenovator Vector System (Qbiogene) according to the manufacturer0s instructions.

Techniques: Immunoprecipitation, Control, Infection, Expressing

Journal: Journal of cellular biochemistry

Article Title: Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

doi: 10.1002/jcb.25430

Figure Lengend Snippet: Fig. 3. Pyk2Y402F does not inhibit migration in Pyk2-KO osteoblasts (OB). Pyk2-KO osteoblasts were infected with adenoviruses expressing Pyk2WT, Pyk2Y402F, or Pyk2K457A. After 3 days, cells were counted and added to migration chambers. The following day, the distance migrated at 0 and 6 h was calculated. (A) Representative images are shown. Dashed lines indicates leading cell edge. Solid bar indicates scale bar at 100 mm. (B) The distance migrated as a percentage of WT osteoblasts is shown. The data shown are average % migration SEM of four experiments. The asterisks indicate statistical significance compared to WT (P < 0.05). (C) Western blotting was used to confirm virus-expressed Pyk2 relative to WT controls. A non-specific protein band seen by the Pyk2 antibody is indicated. Experiments were replicated more than five times. A representative blot is shown which demonstrates higher Pyk2Y402F and lower Pyk2K457A levels compared to Pyk2-KO þ Pyk2WT virus or WT osteoblasts.

Article Snippet: Recombinant adenoviruses expressing WT Pyk2 or its mutants were prepared by recombination of the above plasmids using the Adenovator Vector System (Qbiogene) according to the manufacturer0s instructions.

Techniques: Migration, Infection, Expressing, Western Blot, Virus

Journal: Journal of cellular biochemistry

Article Title: Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

doi: 10.1002/jcb.25430

Figure Lengend Snippet: Fig. 4. Differential regulation of Pyk2 phosphorylation in osteoblasts (OB) by MKs and PMA. (A) Osteoblasts were cultured with MKs for 1, 16, or 24 h. MKs were removed by extensive washing and osteoblasts were treated with 200 nM PMA or vehicle for 1 h. Western blotting was performed for total Pyk2 or phosphorylated Pyk2-Y402 (pY402) as indicated. Densitometry was performed using ImageJ software and used to calculate the ratio of pY402/Pyk2. Studies were replicated three times and representative data are shown. (B) Osteoblasts were co-cultured with MKs for 10 days and assayed for ALP activity. The average standard deviation from four separate osteoblast experiments and eight separate osteoblast þ MKs experiments are shown. In each experiment triplicate cultures were assayed. The asterisk () indicates statistical significance for ALP activity in OB þ MK compared to the OB control.

Article Snippet: Recombinant adenoviruses expressing WT Pyk2 or its mutants were prepared by recombination of the above plasmids using the Adenovator Vector System (Qbiogene) according to the manufacturer0s instructions.

Techniques: Phospho-proteomics, Cell Culture, Western Blot, Software, Activity Assay, Standard Deviation, Control

Journal: Journal of cellular biochemistry

Article Title: Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

doi: 10.1002/jcb.25430

Figure Lengend Snippet: Fig. 5. MKs promote osteoblast migration in the absence of Pyk2. Calvarial osteoblasts (OB) from WT mice or Pyk2-KO mice were plated at a similar density in migration chambers. The next morning, MKs were added to chamber slides (0 h) and osteoblasts were imaged. After 6 h, the wells were re-imaged and the distance traveled in the presence or absence of MKs was calculated and expressed as a percentage of the 0 h control. (A; C) Representative cell images at 0 and 6 h for WT and Pyk2-KO osteoblasts, respectively. The dashed line indicates the leading edge of osteoblasts and solid white bar indicates scale bar (100 mm). (B; D) Data represent the average of three independent studies expressed as a percentage of the genotype-matched control. Although the basal migration of Pyk2-KO is elevated compared to WT osteoblasts (see Fig. 3). MKs significantly increase the migration of both (B) WT osteoblasts and (D) Pyk2-KO osteoblasts to a similar extent. Asterisks () indicate statistical significance for the effects of MKs on WT or Pyk2-KO osteoblasts compared no MKs (P < 0.05).

Article Snippet: Recombinant adenoviruses expressing WT Pyk2 or its mutants were prepared by recombination of the above plasmids using the Adenovator Vector System (Qbiogene) according to the manufacturer0s instructions.

Techniques: Migration, Control