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Effects of nucleotide analogs on the helicase activity of polyomavirus Tags. The helicase activities of the polyomavirus Tags were determined as previously described (83) and measured in the absence or presence of increasing amounts HDP-CDV. The reaction products were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a Fuji FLA-5100 phosphor imager. (A) A representative result. Lanes 1, heat denatured (HD) substrate, and 3, no Tag, served as controls for complete denaturing of the substrate and negative control. Lane 2 presents the unwinding activity of 0.6 µg of SV40 Tag without inhibitor, whereas lanes 4 to 10 show the Tag unwinding activity in the presence of increasing amounts HDP-CDV as indicated. The bands were quantified with <t>ImageQuant</t> analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV. Both curves were fitted using the program GraphPad and the model “log[inhibitor] versus normalized response” plus the equation Y=1001 + 10(X−LogIC50).

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1) Product Images from "Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities"

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01641-20

Figure Legend Snippet: Effects of nucleotide analogs on the helicase activity of polyomavirus Tags. The helicase activities of the polyomavirus Tags were determined as previously described (83) and measured in the absence or presence of increasing amounts HDP-CDV. The reaction products were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a Fuji FLA-5100 phosphor imager. (A) A representative result. Lanes 1, heat denatured (HD) substrate, and 3, no Tag, served as controls for complete denaturing of the substrate and negative control. Lane 2 presents the unwinding activity of 0.6 µg of SV40 Tag without inhibitor, whereas lanes 4 to 10 show the Tag unwinding activity in the presence of increasing amounts HDP-CDV as indicated. The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV. Both curves were fitted using the program GraphPad and the model “log[inhibitor] versus normalized response” plus the equation Y=1001 + 10(X−LogIC50).

Techniques Used: Activity Assay, Negative Control, Software, Standard Deviation, Inhibition

ssDNA and dsDNA binding activities for SV40 Tag. The binding of SV40 Tag to ssDNA and dsDNA was analyzed as previously described (51) to optimize Tag concentrations required to investigate the inhibition of Tag-DNA interactions by nucleotide analogs. (A and C) The binding products using (A) ssDNA and (B) SV40 Tag site 2-containing dsDNA were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager. The bands were quantified with ImageQuant analysis software, and the bound DNA was calculated by determining the ratio of the bound to total DNA. The data were fitted using GraphPad and the model “one site-specific binding with Hill slope” to obtain the binding constants (panels B and D). The KD values for Tag binding to ssDNA and dsDNA are 131.9 ± 3.1 nM and 120.8 ± 4.7 nM, respectively.

Figure Legend Snippet: ssDNA and dsDNA binding activities for SV40 Tag. The binding of SV40 Tag to ssDNA and dsDNA was analyzed as previously described (51) to optimize Tag concentrations required to investigate the inhibition of Tag-DNA interactions by nucleotide analogs. (A and C) The binding products using (A) ssDNA and (B) SV40 Tag site 2-containing dsDNA were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager. The bands were quantified with ImageQuant analysis software, and the bound DNA was calculated by determining the ratio of the bound to total DNA. The data were fitted using GraphPad and the model “one site-specific binding with Hill slope” to obtain the binding constants (panels B and D). The KD values for Tag binding to ssDNA and dsDNA are 131.9 ± 3.1 nM and 120.8 ± 4.7 nM, respectively.

Techniques Used: Binding Assay, Inhibition, Software

Effects of HDP-CDV on the DNA binding activity of SV40 Tag. Following the determination of the binding constants for SV40 Tag binding to ssDNA and dsDNA, the effects of HDP-CDV on ssDNA and dsDNA binding function of Tag were determined via electrophoretic mobility shift assay (EMSA) at the indicated analog concentrations in the presence of 300 nM Tag. The reaction Tag-ssDNA and Tag-dsDNA products were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager. (A and C) Representative results. The bands were quantified with ImageQuant software, and the relative DNA binding was calculated by determining the percentage ratio of bound DNA of the HDP-CDV treated samples to the solvent-treated sample. (B and D) The mean and standard deviation from 3 independent experiments are presented. The data were fitted with the model “log[inhibitor] versus response − variable slope” on GraphPad to obtain the binding curves and inhibition constants using the equations Y=−1.09 + 92.81 + 10(X−LogIC50)*3.406 and Y=−7.83 + 96.51 + 10(X−LogIC50)*2.129 (panels B and D, respectively). The IC50 values for the inhibition of binding of Tag to ssDNA and dsDNA by HDP-CDV were determined as 17.4 ± 1.02 µM and 22.1 ± 1.04 µM, respectively.

Figure Legend Snippet: Effects of HDP-CDV on the DNA binding activity of SV40 Tag. Following the determination of the binding constants for SV40 Tag binding to ssDNA and dsDNA, the effects of HDP-CDV on ssDNA and dsDNA binding function of Tag were determined via electrophoretic mobility shift assay (EMSA) at the indicated analog concentrations in the presence of 300 nM Tag. The reaction Tag-ssDNA and Tag-dsDNA products were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager. (A and C) Representative results. The bands were quantified with ImageQuant software, and the relative DNA binding was calculated by determining the percentage ratio of bound DNA of the HDP-CDV treated samples to the solvent-treated sample. (B and D) The mean and standard deviation from 3 independent experiments are presented. The data were fitted with the model “log[inhibitor] versus response − variable slope” on GraphPad to obtain the binding curves and inhibition constants using the equations Y=−1.09 + 92.81 + 10(X−LogIC50)*3.406 and Y=−7.83 + 96.51 + 10(X−LogIC50)*2.129 (panels B and D, respectively). The IC50 values for the inhibition of binding of Tag to ssDNA and dsDNA by HDP-CDV were determined as 17.4 ± 1.02 µM and 22.1 ± 1.04 µM, respectively.

Techniques Used: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Software, Standard Deviation, Inhibition

HDP-CDV does not influence the DNA binding activity of RPA. (A and B) The effects of HDP-CDV on the binding of RPA to ssDNA were determined via EMSA at the indicated analog concentrations in the presence of 2 nM RPA. The free ssDNA and the RPA-ssDNA complexes were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager (panel A). The bands were quantified with ImageQuant software, and the relative DNA binding was calculated by determining the percentage ratio of bound DNA of the HDP-CDV-treated samples to the solvent-treated sample. Then the results from 3 independent experiments were fitted with various nonlinear regression models using GraphPad, but none yielded reliable results. Therefore, a linear regression analysis was performed yielding the trendline with the equation y = − 3.5114×112.6; r2 = 0.9776 (panel B).

Figure Legend Snippet: HDP-CDV does not influence the DNA binding activity of RPA. (A and B) The effects of HDP-CDV on the binding of RPA to ssDNA were determined via EMSA at the indicated analog concentrations in the presence of 2 nM RPA. The free ssDNA and the RPA-ssDNA complexes were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager (panel A). The bands were quantified with ImageQuant software, and the relative DNA binding was calculated by determining the percentage ratio of bound DNA of the HDP-CDV-treated samples to the solvent-treated sample. Then the results from 3 independent experiments were fitted with various nonlinear regression models using GraphPad, but none yielded reliable results. Therefore, a linear regression analysis was performed yielding the trendline with the equation y = − 3.5114×112.6; r2 = 0.9776 (panel B).

Techniques Used: Binding Assay, Activity Assay, Software

Software:

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities
Article Snippet: .. The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV. .. Both curves were fitted using the program GraphPad and the model “log[inhibitor] versus normalized response” plus the equation Y = 100 1 + 10 ( X − LogIC 50 ) .

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities
Article Snippet: The plate was dried and exposed to phosphor imager screens and subsequently analyzed using a Fujifilm FLA-5100 imager. .. The bands were quantitated using ImageQuant software (Fujifilm). ..

Article Title: Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1
Article Snippet: The immunoreactive bands were visualized using a Clarity MAX Western ECL substrate (Bio-Rad, Hercules, CA, USA) and ImageQuant LAS-4000 (Cytiva, Tokyo, Japan). .. The relative densities of the bands were quantified using Image QuantTL software (FUJIFILM Corporation, Tokyo, Japan). ..

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities
Article Snippet: The plate was dried and exposed to phosphor imager screens, and 261 subsequently analyzed using Fujifilm FLA-5100. .. The bands were quantitated using 262 image quant software (Fujifilm). ..

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities
Article Snippet: The gel was exposed for autoradiography to Fuji imager screens at −20°C and subsequently developed using a Fujifilm FLA-5100 imager. .. The band signals were quantitated using ImageQuant software (Fujifilm). ..

Solvent:

Standard Deviation:

Inhibition:

other:

Article Title: Sequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7
Article Snippet: Signal from scanned phosphor imager screens was quantified in the software ImageQuant (Fujifilm). re p o rtfo lio | rep o rtin g su m m ary A pril2023 Data Policy information about availability of data All manuscripts must include a data availability statement.

Article Title: Functional genomics of primary congenital glaucoma by pathway analysis and functional characterization of CYP1B1 mutations.
Article Snippet: CYP1B1 is the most common gene implicated in primary congenital glaucoma (PCG) − the most common form of childhood glaucoma.. How CYP1B1 mutations cause PCG is not known.. Understanding the mechanism of PCG caused by CYP1B1 mutations is crucial for disease management, therapeutics development, and potential prevention.

Image Search Results

Phosphorylation of MSK1 via the p38 kinase pathway upon BMP-6 stimulation in ATDC5 cells. A , phosphorylation of MSK1 (Ser376) and MSK1 (Ser360) by BMP-6. ATDC5 cells were stimulated with 25 ng/ml BMP-6 for the indicated times. The total cell lysates were then used for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-Smad1/5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6. B , Inhibition of BMP-6-induced MSK1 phosphorylation by dorsomorphin, a BMP type I receptor kinase inhibitor. ATDC5 cells were pretreated with 10 μM dorsomorphin for 1 h. Thereafter, cells were stimulated with 25 ng/ml BMP-6 for the indicated times. The total cell lysates were used for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of dorsomorphin. C , Involvement of the ERK pathway in the phosphorylation of MSK1 by BMP-6. ATDC5 cells were pretreated with 20 μM U0126 1 h before stimulation with 25 ng/ml BMP-6 for the indicated times. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of U0126. D , decrease in BMP-6-induced phosphorylation of MSK1 in the presence of the p38 kinase inhibitor. ATDC5 cells pretreated with 10 μM SB203580, a p38 kinase inhibitor, for 1 h were stimulated with 25 ng/ml BMP-6 for the indicated times. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of SB203580. A-D , white and black arrowheads indicate phospho-MSK1 (Ser376)-specific and nonspecific bands, respectively. E , decrease in BMP-6-mediated phospho-CREB (Ser133) expression by the MSK1 kinase inhibitor, SB747651A. ATDC5 cells were cultured with 10 μM SB747651A for 1 h. Prior to preparation of the total lysates, 25 ng/ml BMP-6 was added to the culture media for the indicated times. The total expression levels of phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth , and bottom panels , respectively. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of SB747651A. F , chondrogenic differentiation of ATDC5 cells by BMP-6 in the presence of kinase inhibitors. ATDC5 cells cultured at a high density were stimulated with 25 ng/ml BMP-6 in the presence (+) or absence (−) of kinase inhibitors (20 μM U0126, 10 μM SB203580, 10 μM SB747651A, or 10 μM dorsomorphin). Two weeks later, the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the 1-h treatment of cells with BMP-6 alone. G , representative images of ex vivo chondrogenic differentiation of mouse metatarsal bones induced by BMP-6 in the presence of kinase inhibitors. Each mouse metatarsal bone was cultured with or without 25 ng/ml BMP-6 for 7 days, either in the absence or presence of kinase inhibitors (20 μM U0126, 10 μM SB203580, 10 μM SB747651A, or 10 μM dorsomorphin). Then, each bone was stained with Alcian blue and Alizarin Red S. Two representative images are shown in each treatment. a , proliferative chondrocytes; b , clear zone represented as hypertrophic chondrocytes; c , chondrocyte matrix calcified by mature hypertrophic chondrocytes stained with alizarin red S. H , relative area of chondrocyte differentiation in mouse metatarsal bones induced by BMP-6 in the presence of kinase inhibitors. The total areas of chondrocyte differentiation (a + b + c) were measured. The total areas of chondrocyte differentiation (a + b + c) were measured using ImageJ. Asterisks indicate significant differences. BMP, bone morphogenetic protein; CREB, cAMP-response element-binding protein; MSK, mitogen- and stress-activated protein kinase; ND; not determined.

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: Phosphorylation of MSK1 via the p38 kinase pathway upon BMP-6 stimulation in ATDC5 cells. A , phosphorylation of MSK1 (Ser376) and MSK1 (Ser360) by BMP-6. ATDC5 cells were stimulated with 25 ng/ml BMP-6 for the indicated times. The total cell lysates were then used for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-Smad1/5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6. B , Inhibition of BMP-6-induced MSK1 phosphorylation by dorsomorphin, a BMP type I receptor kinase inhibitor. ATDC5 cells were pretreated with 10 μM dorsomorphin for 1 h. Thereafter, cells were stimulated with 25 ng/ml BMP-6 for the indicated times. The total cell lysates were used for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of dorsomorphin. C , Involvement of the ERK pathway in the phosphorylation of MSK1 by BMP-6. ATDC5 cells were pretreated with 20 μM U0126 1 h before stimulation with 25 ng/ml BMP-6 for the indicated times. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of U0126. D , decrease in BMP-6-induced phosphorylation of MSK1 in the presence of the p38 kinase inhibitor. ATDC5 cells pretreated with 10 μM SB203580, a p38 kinase inhibitor, for 1 h were stimulated with 25 ng/ml BMP-6 for the indicated times. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of SB203580. A-D , white and black arrowheads indicate phospho-MSK1 (Ser376)-specific and nonspecific bands, respectively. E , decrease in BMP-6-mediated phospho-CREB (Ser133) expression by the MSK1 kinase inhibitor, SB747651A. ATDC5 cells were cultured with 10 μM SB747651A for 1 h. Prior to preparation of the total lysates, 25 ng/ml BMP-6 was added to the culture media for the indicated times. The total expression levels of phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth , and bottom panels , respectively. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of SB747651A. F , chondrogenic differentiation of ATDC5 cells by BMP-6 in the presence of kinase inhibitors. ATDC5 cells cultured at a high density were stimulated with 25 ng/ml BMP-6 in the presence (+) or absence (−) of kinase inhibitors (20 μM U0126, 10 μM SB203580, 10 μM SB747651A, or 10 μM dorsomorphin). Two weeks later, the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the 1-h treatment of cells with BMP-6 alone. G , representative images of ex vivo chondrogenic differentiation of mouse metatarsal bones induced by BMP-6 in the presence of kinase inhibitors. Each mouse metatarsal bone was cultured with or without 25 ng/ml BMP-6 for 7 days, either in the absence or presence of kinase inhibitors (20 μM U0126, 10 μM SB203580, 10 μM SB747651A, or 10 μM dorsomorphin). Then, each bone was stained with Alcian blue and Alizarin Red S. Two representative images are shown in each treatment. a , proliferative chondrocytes; b , clear zone represented as hypertrophic chondrocytes; c , chondrocyte matrix calcified by mature hypertrophic chondrocytes stained with alizarin red S. H , relative area of chondrocyte differentiation in mouse metatarsal bones induced by BMP-6 in the presence of kinase inhibitors. The total areas of chondrocyte differentiation (a + b + c) were measured. The total areas of chondrocyte differentiation (a + b + c) were measured using ImageJ. Asterisks indicate significant differences. BMP, bone morphogenetic protein; CREB, cAMP-response element-binding protein; MSK, mitogen- and stress-activated protein kinase; ND; not determined.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Phospho-proteomics, Western Blot, Expressing, Inhibition, Cell Culture, Staining, Ex Vivo, Binding Assay

I nflu ence of MSK1 deficiency on BMP-6–mediated chondrogenic differentiation. A , expression of MSK1 in ATDC5 cells. Total lysates from each transformant were prepared for Western blot analyses. The total expression levels of MSK1 and β-actin are indicated in the upper and lower panels , respectively. White and black arrowheads indicate MSK1-specific and nonspecific bands, respectively. The intensity of the band for MSK1 was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to cells carrying shControl. B , chondrogenic differentiation of ATDC5 cells with decreased expression of MSK1 in the presence of BMP-6. The cells were seeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying shControl with BMP-6 for 14 days. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; qPCR, quantitative PCR.

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: I nflu ence of MSK1 deficiency on BMP-6–mediated chondrogenic differentiation. A , expression of MSK1 in ATDC5 cells. Total lysates from each transformant were prepared for Western blot analyses. The total expression levels of MSK1 and β-actin are indicated in the upper and lower panels , respectively. White and black arrowheads indicate MSK1-specific and nonspecific bands, respectively. The intensity of the band for MSK1 was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to cells carrying shControl. B , chondrogenic differentiation of ATDC5 cells with decreased expression of MSK1 in the presence of BMP-6. The cells were seeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying shControl with BMP-6 for 14 days. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Expressing, Western Blot, Staining, Real-time Polymerase Chain Reaction

R equi rement of N-terminal and C-terminal kinase activities of MSK1 for the induction of chondrocyte differentiation. A , depiction of MSK1 and its mutants used in . B , phosphorylation of MSK1 or its mutants in ATDC5 cells stimulated with BMP-6. To induce the expression of ectopic MSK1 or its mutants in ATDC5 cells, cells were cultured in the presence of tetracycline for 16 h. Thereafter, the ATDC5 transformants were stimulated with 25 ng/ml BMP-6 for 1 h. The total expression levels of phospho-MSK1 (Ser376), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensities of the bands for phospho-MSK1 (Ser376) and phospho-CREB (Ser133) were normalized to the intensities of the bands corresponding to MSK1 and β-actin, respectively. The relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6 alone. C , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or its mutants in the presence of BMP-6. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were reseeded at a high density and stimulated with 25 ng/ml BMP-6. Seven ( upper photos ) or 14 days later ( lower photos ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 alone for 14 days. D and E , According to C , ATDC5 cells expressing WT MSK1 or MSK1 (D195A/D565A) were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, the total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( D ) and collagen10a1 mRNAs ( E ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; CTKD, C-terminal kinase domain; HM, hydrophobic motif; MSK, mitogen- and stress-activated protein kinase; ND, not determined; NTKD, N-terminal kinase domain; qPCR, quantitative PCR.

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: R equi rement of N-terminal and C-terminal kinase activities of MSK1 for the induction of chondrocyte differentiation. A , depiction of MSK1 and its mutants used in . B , phosphorylation of MSK1 or its mutants in ATDC5 cells stimulated with BMP-6. To induce the expression of ectopic MSK1 or its mutants in ATDC5 cells, cells were cultured in the presence of tetracycline for 16 h. Thereafter, the ATDC5 transformants were stimulated with 25 ng/ml BMP-6 for 1 h. The total expression levels of phospho-MSK1 (Ser376), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensities of the bands for phospho-MSK1 (Ser376) and phospho-CREB (Ser133) were normalized to the intensities of the bands corresponding to MSK1 and β-actin, respectively. The relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6 alone. C , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or its mutants in the presence of BMP-6. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were reseeded at a high density and stimulated with 25 ng/ml BMP-6. Seven ( upper photos ) or 14 days later ( lower photos ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 alone for 14 days. D and E , According to C , ATDC5 cells expressing WT MSK1 or MSK1 (D195A/D565A) were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, the total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( D ) and collagen10a1 mRNAs ( E ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; CTKD, C-terminal kinase domain; HM, hydrophobic motif; MSK, mitogen- and stress-activated protein kinase; ND, not determined; NTKD, N-terminal kinase domain; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction

M SK1 m utants lacking phosphorylation site targeted by p38 kinase and C-terminal kinase of MSK1. A , depiction of MSK1 and its mutants used in and . B , phosphorylation of MSK1 or its mutants in ATDC5 cells stimulated with BMP-6. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were stimulated with 25 ng/ml BMP-6 for 1 h and their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. C , comparison between serine-360 and threonine-581 in MSK1 as targeted amino acid residues for p38 kinase. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were stimulated with 25 ng/ml BMP-6 for 1 h and then their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. D , phosphorylation of threonine at position 581 in MSK1 upon BMP-6 stimulation. As described in B , cell lysates were prepared from each transformant. The total expression levels of phospho-MSK1 (Thr581), MSK1, phospho-Smad1/5, and β-actin are indicated in the upper, second, third, and bottom panels , respectively. The intensity of the band for phospho-MSK1 (T581) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. E , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or its mutants in the presence of BMP-6. After the expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in C , the cells were reseeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 alone for 14 days. F and G , expression of aggrecan and collagen 10a1 mRNAs in ATDC5 cells and their transformants. According to , D and E , ATDC5 cells expressing WT MSK1 or its mutants were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( F ) and collagen10a1 mRNAs ( G ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; CREB, cAMP-response element-binding protein; MSK, mitogen- and stress-activated protein kinase; ND, not determined; qPCR, quantitative PCR.

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: M SK1 m utants lacking phosphorylation site targeted by p38 kinase and C-terminal kinase of MSK1. A , depiction of MSK1 and its mutants used in and . B , phosphorylation of MSK1 or its mutants in ATDC5 cells stimulated with BMP-6. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were stimulated with 25 ng/ml BMP-6 for 1 h and their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. C , comparison between serine-360 and threonine-581 in MSK1 as targeted amino acid residues for p38 kinase. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were stimulated with 25 ng/ml BMP-6 for 1 h and then their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. D , phosphorylation of threonine at position 581 in MSK1 upon BMP-6 stimulation. As described in B , cell lysates were prepared from each transformant. The total expression levels of phospho-MSK1 (Thr581), MSK1, phospho-Smad1/5, and β-actin are indicated in the upper, second, third, and bottom panels , respectively. The intensity of the band for phospho-MSK1 (T581) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. E , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or its mutants in the presence of BMP-6. After the expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in C , the cells were reseeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 alone for 14 days. F and G , expression of aggrecan and collagen 10a1 mRNAs in ATDC5 cells and their transformants. According to , D and E , ATDC5 cells expressing WT MSK1 or its mutants were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( F ) and collagen10a1 mRNAs ( G ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; CREB, cAMP-response element-binding protein; MSK, mitogen- and stress-activated protein kinase; ND, not determined; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Phospho-proteomics, Expressing, Western Blot, Comparison, Staining, Cell Culture, Binding Assay, Real-time Polymerase Chain Reaction

Req u irement of autophosphorylation of Ser 376 in MSK1 for its kinase activity upon BMP-6 stimulation. A , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or MSK1 (S376A) mutant in the presence of BMP-6. After the expression of WT MSK1 or MSK1 (S376A) mutant in ATDC5 cells using tetracycline as described in C , the cells were reseeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 for 14 days. B and C , expression of aggrecan and collagen10a1 mRNAs in ATDC5 cells and their transformant. According to , D and E , ATDC5 cells expressing the WT MSK1 or its mutant were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( B ) and collagen10a1 mRNAs ( C ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; qPCR, quantitative PCR.

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: Req u irement of autophosphorylation of Ser 376 in MSK1 for its kinase activity upon BMP-6 stimulation. A , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or MSK1 (S376A) mutant in the presence of BMP-6. After the expression of WT MSK1 or MSK1 (S376A) mutant in ATDC5 cells using tetracycline as described in C , the cells were reseeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 for 14 days. B and C , expression of aggrecan and collagen10a1 mRNAs in ATDC5 cells and their transformant. According to , D and E , ATDC5 cells expressing the WT MSK1 or its mutant were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( B ) and collagen10a1 mRNAs ( C ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Activity Assay, Mutagenesis, Expressing, Staining, Cell Culture, Real-time Polymerase Chain Reaction

Effect of the BMP/Smad-dependent pathway on BMP-6–mediated chondrogenic differentiation. A , expression of Smad4 in ATDC5 cells. Total lysates from each transformant were prepared for Western blot analyses. The total expression levels of Smad4 and β-actin are indicated in the upper and lower panels , respectively. The intensity of the band for Smad4 was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to cells carrying shControl. B , chondrogenic differentiation of ATDC5 cells with decreased expression of Smad4 in the presence of BMP-6. The cells were seeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying shControl with BMP-6 for 14 days. C and D , expression of aggrecan and collagen10a1 mRNA in ATDC5 cells with Smad4 knockdown. According to <xref ref-type=

Fig. 2 , D and E , ATDC5 cells with decreased expression of Smad4 were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( C ) and collagen10a1 mRNAs ( D ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. E , effect of Smad4 deficiency on BMP-6–induced p38 phosphorylation. The cells were stimulated with 25 ng/ml BMP-6 for 1 h and then their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), MSK1, phospho-p38 (Thr180/Tyr182), phospho-Smad1/5, and β-actin are indicated in the upper, second, third, fourth, and bottom panels , respectively. The intensity of the band for phospho-MSK1 (S376) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells carrying shControl with BMP-6. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; ND, not determined; qPCR, quantitative PCR. " width="100%" height="100%">

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: Effect of the BMP/Smad-dependent pathway on BMP-6–mediated chondrogenic differentiation. A , expression of Smad4 in ATDC5 cells. Total lysates from each transformant were prepared for Western blot analyses. The total expression levels of Smad4 and β-actin are indicated in the upper and lower panels , respectively. The intensity of the band for Smad4 was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to cells carrying shControl. B , chondrogenic differentiation of ATDC5 cells with decreased expression of Smad4 in the presence of BMP-6. The cells were seeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying shControl with BMP-6 for 14 days. C and D , expression of aggrecan and collagen10a1 mRNA in ATDC5 cells with Smad4 knockdown. According to Fig. 2 , D and E , ATDC5 cells with decreased expression of Smad4 were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( C ) and collagen10a1 mRNAs ( D ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. E , effect of Smad4 deficiency on BMP-6–induced p38 phosphorylation. The cells were stimulated with 25 ng/ml BMP-6 for 1 h and then their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), MSK1, phospho-p38 (Thr180/Tyr182), phospho-Smad1/5, and β-actin are indicated in the upper, second, third, fourth, and bottom panels , respectively. The intensity of the band for phospho-MSK1 (S376) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells carrying shControl with BMP-6. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; ND, not determined; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Expressing, Western Blot, Staining, Knockdown, Cell Culture, Phospho-proteomics, Real-time Polymerase Chain Reaction

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