vx702  (LC Laboratories)

Journal: MedComm

Article Title: Targeting the Cdc2‐like kinase 2 for overcoming platinum resistance in ovarian cancer

doi: 10.1002/mco2.537

Figure Lengend Snippet: Cisplatin induces CLK2 upregulation via p38. (A) Western blot analysis of CLK2 expression in A2780 cells treated with various signaling pathway inhibitors or dimethyl sulfoxide (DMSO) in the presence or absence of cisplatin. Only p38 inhibitor attenuated the expression of CLK2. (B) CLK2 mRNA level in A2780 cells treated with cisplatin in the presence or absence of p38 inhibitor was measured by qRT‐PCR. Data was shown as mean ± SD. p Values were determined by Student's t ‐test. (C) A2780 cells treated with phosphate‐buffered saline (PBS), or cisplatin, or cisplatin plus p38 inhibitor (p38i), or cisplatin and p38 overexpression (p38‐Myc) were incubated with 20 µg/mL cycloheximide (CHX) for the indicated periods and then analyzed by Western blot. The results were normalized to the levels of GAPDH. (D) Quantitation of CLK2 protein levels was based on the Western blot result. Data were shown as mean ± SD. (E) Coimmunoprecipitation assay showing the presence of a complex containing CLK2 and p38. CLK2 antibody coprecipitating p38 (top panel). p38 antibody coprecipitating CLK2 (bottom panel). Input, protein expression in cell lysates detected by Western blot. IgG, negative control. IP, expression of compound coprecipitated by CLK2 or p38 antibody. n.s., no significance.

Article Snippet: To determine the half‐life of CLK2, A2780 cells were treated either with PBS, cisplatin (6.67 μM) or p38i (10 μM, VX702, #A8687; APExBio) plus cisplatin.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Saline, Over Expression, Incubation, Quantitation Assay, Co-Immunoprecipitation Assay, Negative Control

Journal: MedComm

Article Title: Targeting the Cdc2‐like kinase 2 for overcoming platinum resistance in ovarian cancer

doi: 10.1002/mco2.537

Figure Lengend Snippet: p38 and ring finger protein 8 (RNF8) mediate CLK2 protein ubiquitination. (A) Western blot analyses of CLK2 expression in A2780 and Skov3 cells which were treated with cisplatin (16.7 µM) in the presence or absence of p38 inhibitor (10 µM). Cells were incubated with MG132 (10 µM) for the indicated periods. (B) Ubiquitination assays of CLK2 in human embryonic kidney (HEK293T) cells transfected with Flag‐CLK2, Myc‐p38, and HA‐ubiquitin or its mutants (K48R, K48o, K63R, K63o) plasmids. IP, expression of compound coprecipitated by Flag antibody. WCL, whole cell lysate. (C) Ubiquitination assays of CLK2 in HEK293T cells transfected with Myc‐p38, HA‐Ub‐K63o, and CLK2‐WT or its mutants (T343A, K192R) plasmids. IP, expression of compound coprecipitated by CLK2 antibody. WCL, whole cell lysate. (D) Coimmunoprecipitation assay showing the endogenous (top panel) presence of a complex containing CLK2 and RNF8 and the exogenous (bottom panel) presence of a complex containing CLK2 and Flag‐RNF8. Input, protein expression in cell lysates detected by Western blot. IgG, negative control. IP, expression of compound coprecipitated by CLK2, RNF8, or Flag antibody. WCL, whole cell lysate. (E) Ubiquitination assays of CLK2 in HEK293T cells transfected with CLK2, Flag‐RNF8, and HA‐ubiquitin or its mutants (K48o, K63o) plasmids. IP, expression of compound coprecipitated by CLK2 antibody. WCL, whole cell lysate. (F) Ubiquitination assays of CLK2 in HEK293T cells cotransfected by various combinations of plasmids expressing CLK2, Myc‐p38, Flag‐RNF8, and HA‐Ub‐K63o. IP, expression of compound coprecipitated by CLK2 antibody; WCL, whole cell lysate.

Article Snippet: To determine the half‐life of CLK2, A2780 cells were treated either with PBS, cisplatin (6.67 μM) or p38i (10 μM, VX702, #A8687; APExBio) plus cisplatin.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Incubation, Transfection, Co-Immunoprecipitation Assay, Negative Control

Journal: PLoS ONE

Article Title: Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association

doi: 10.1371/journal.pone.0183343

Figure Lengend Snippet: (A) Serum growth factors do not affect the phosphorylation status of p130cas. Cells were serum starved (in the presence or absence of ouabain) for 24 hrs after which serum was added for indicated times. Cell extracts were prepared at 7 min and 1 hr after serum addition, and analyzed by western blotting using 4G10 monoclonal antibody. The level of tyrosine phosphorylation of p130cas was measured using Image J. Control, untreated starved cells; Ouabain, starved cells pretreated with 200 nM ouabain; 7m, serum added for 7 min to untreated starved cells; O7m, serum added for 7 min to starved cells pretreated with ouabain; 1 hr, serum added for 1 hr to untreated starved cells; O1h, serum added for 1 hr cells to starved cells pretreated with ouabain. The level of tyrosine phosphorylation of p130cas in untreated cells was set at 100%. Quantitation was used to calculate statistically significant differences between groups, indicated with a * (P<0.05). (B) To analyze an effect of protein kinases on phosphorylation of p130cas, cells were treated with 500 nM ouabain or with 5 μM of inhibitors of the indicated protein kinases for 24 hrs, proteins extracted and the level of tyrosine phosphorylation of p130cas was measured as described above. The inhibitors used were: Src inhibitor AZD05030, MEK inhibitor PD0325901, PI3K inhibitor TGX221 and p38MAPK inhibitor VX702. The level of tyrosine phosphorylation of p130cas was set at 100% for untreated cells. Quantitation was used to calculate statistically significant differences between groups, indicated with a * (P<0.05). (C) Western blot analysis of cells treated with combinations of 500 nM ouabain and protein kinase inhibitors for 24 hrs. Upper panel: blots were probed with 4G10 monoclonal antibody; lower panel, blots were probed with anti-actin antibody. Cells were untreated (lane 1) or treated with ouabain (lane 2), Src inhibitor AZD05030 (lane 3), ouabain plus Src inhibitor AZD05030 (lane 4), MEK inhibitor PD0325901 (lane 5), ouabain plus MEK inhibitor PD0325901 (lane 6), p38MAPK inhibitor VX702 (lane 7), and ouabain plus p38MAPK inhibitor VX702 (lane 8). (D) To detect Src-Na,K-ATPase interaction, cells were ouabain treated (200 nM for 48 hrs) and extracts were analyzed by western blotting with anti- Na,K-ATPase antibody directly (lanes 1 and 4) or after immunoprecipitation using non-specific mouse IgG antibody (lanes 2 and 3) or anti-Src monoclonal antibody (lanes 5 and 6). Upper panel: lane 1, whole extract; lane 2, Na,K-ATPase bound to non-specific mouse IgG; lane 3, Na,K-ATPase not bound to non-specific mouse IgG; lane 4, whole extract; lane 5, Na,K-ATPase bound to Src; lane 6, Na,K-ATPase not bound to Src. The lower panel shows western blot controls using anti-actin antibody. (E) Western blot analysis of Src in untreated cells (lane 1) and cells treated with ouabain (200 nM for 48 hrs) (lane 2). Top panel (Pi-Y416), western blotting using anti-phospho tyrosine-416 Src antibody. Lower panel (total Src), western blotting using anti-Src antibody to detect total Src protein.

Article Snippet: Src inhibitor AZD0530, MEK inhibitor PD0325901, and p38MAPK inhibitor VX702 were purchased from Selleckchem (Houston, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Quantitation Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

doi: 10.1038/ncomms6413

Figure Lengend Snippet: ( a ) Western blot analysis of total and phospho-eIF4E1(S209) as well as total and phospho-ERK after 24 h treatment with an MEK inhibitor AZD6244 (AZD 200 nM), compared with untreated and vehicle controls in HLY-1, GM02184 and Pfeiffer cell lines. Densitometry analysis is shown in . ( b ) Western blot analysis of phospho-MNKs (antibody detects p-MNK1 and p-MNK2), total MNK1, total MNK2, total and phospho-eIF4E1(S209) after 1 h treatment with a p38 inhibitor VX702 (200 nM). Densitometry analysis is shown in . ( c ) Western blot analysis of total and phospho-eIF4E1 and MCL-1 post 4 h treatment with a p38 inhibitor VX702. ( d ) Densitometry analysis of phospho-eIF4E1(S209) and ( e ) MCL-1 band intensity of western blot in , relative to GAPDH following 4 h treatment with vehicle (black bar) or 200 nM VX702 (white bar). Mean±s.d., n =3, * P -value of Student’s t -test <0.05. ( f ) Trypan blue exclusion assay of HLY-1, GM02184 and Pfeiffer cells after 72 h treatment with vehicle (black bar) or 200 nM VX702 (white bar). Mean±s.d., n =3, * P -value of Student’s t -test <0.001. ( g ) Representative CFSE analysis of HLY-1 cells 48 and 72 h post treatment with 200 nM VX702 (blue line) or vehicle (DMSO; red line). P0 denotes initial population, while P1, P2 and P3 denote subsequent daughter cell populations. Three independent experiments are shown in . ( h ) Western blot showing total and phospho-eIF4E1(S209) as well as MCL-1 with or without 4 h treatment with 200 nM VX702 in HLY-1 cells expressing empty vector (EV), MNK1-wildtype (M1WT), MNK1-phosphomimetic (M1TD), MNK1-phosphonull (M1 AA), and MNK2-wildtype (M2WT). ( i , j ) Densitometry analysis showing relative band intensity of ( i ) phospho-eIF4E1(S209) or ( j ) MCL-1 to GAPDH in HLY-1 cells treated with vehicle (black bar) or 200 nM VX702 (white bar) for 4 h of immunoblot in (mean±s.d., n =3, * P -value of Student’s t -test <0.05). ( k ) Summarizing figure illustrating the p38-regulated MNK-dependent eIF4E1 phosphorylation in DLBCL cells. Cartoon depicts two potential modes of disrupting the p38-MNK-eIF4E1 axis in DLBCL, that are, via the use of p38 or MNK inhibitors. Full immunoblots are shown in .

Article Snippet: VX702 (p38 inhibitor) was purchased from Cayman Chemical, and MEK inhibitor AZD6244 was purchased from CalBiochem.

Techniques: Western Blot, Trypan Blue Exclusion Assay, Expressing, Plasmid Preparation, Phospho-proteomics

Journal: Nature Communications

Article Title: MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

doi: 10.1038/ncomms6413

Figure Lengend Snippet: ( a ) On activation by p38, MNKs phosphorylate eIF4E1 at S209. ( b ) In the absence of MNK kinase activity, eIF4E1 cannot be phosphorylated, while in the ( c ) absence of MNK protein expression or its physical suppression, eIF4E1 protein expression is downregulated. ( d ) The unphosphorylated eIF4E1 (at S209) form is stimulatory for eIF4E3 protein upregulation. Increased abundance of eIF4E3 in a cellular context enhances the ability for eIF4E3 to bind cap. The relative abundance of either eIF4E1 or eIF4E3 is determined by MNKs. The accessibility to mRNA cap structure by both eIF4Es mandates a distinct cellular translatome that dictates pro- or anti-oncogenic phenotype.

Article Snippet: VX702 (p38 inhibitor) was purchased from Cayman Chemical, and MEK inhibitor AZD6244 was purchased from CalBiochem.

Techniques: Activation Assay, Activity Assay, Expressing