rsk2 sc 9986 mouse  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Endogenous LARG phosphorylation at S1288 and RhoA activation in GBM tissues . A , the phospho-LARG S1288 specific antibody was tested in U87MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min in the presence or absence of BI-D1870. B , the phospho-LARG S1288 antibody demonstrates specificity to the phosphorylated form of the protein. U87MG cells were transfected with Myc-LARG-WT of Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min. Protein was harvested and lysates were treated with λ phosphatase prior to immunoblotting. C , RSK2 phosphorylates LARG at S1288 in PDX-derived GBM39 cells. Cells maintained in complete medium with 10% FBS were treated with BI-D1870 for 4 h. Subsequently cells were starved in 0.1% FBS medium for 24 h prior to 10 min EGF treatment alone or after prior 4 h BID treatment. Immunoblotting was used to determine total expression and phosphorylation of target proteins. The results are representative of three independent experiments. D , levels of activated RhoA-GTP positively correlate with levels of phosphorylated LARG S1288 in human GBM tumor lysates. Endogenous total LARG, pLARG-S1288 and RhoA expression in seven human GBM tumor lysates were analyzed by immunoblotting. Activated RhoA-GTP was isolated by immunoprecipitation and detected with a RhoA antibody. Signals for each target protein expression were normalized to corresponding sample β-tubulin expression. A linear regression analysis was performed to assess the relationship between Activated RhoA expression and pLARG S1288 expression in all seven tumors.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Activation Assay, Transfection, Western Blot, Derivative Assay, Expressing, Isolation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Model of LARG activation of RhoA in response to RSK2 phosphorylation . Schematic of the proposed signaling mechanism in which EGF stimulation activates RSK2, which leads to the phosphorylation of LARG at serine 1288. Phosphorylation of LARG at serine 1288 then promotes plasma membrane localization of LARG and binding to RhoA and subsequent RhoA activation.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Binding Assay

Journal: Molecular Cancer

Article Title: miR-634 restores drug sensitivity in resistant ovarian cancer cells by targeting the Ras-MAPK pathway

doi: 10.1186/s12943-015-0464-4

Figure Lengend Snippet: MiR-634 regulates genes involved in cell cycle regulation and the Ras-MAPK pathway. Ovarian cancer cell lines (A2780, A2780 DDP) and patient derived tumor cells (culture #3) were transiently transfected with scrambled control (-) or miR-634 mimic (+). 48 h after transfection protein lysates were analyzed for the expression of putative miR-634 target genes by Western blotting (A, B) or RT-PCR (C). a Transfection with miR-634 reduces Cyclin D1 protein levels in the A2780 cell lines and in primary ovarian cancer cell cultures. β-actin is used as a loading control. b MiR-634 overexpression reduces protein levels of GRB2, ERK2, RSK1 and RSK2, components of the Ras-MAPK pathway in the A2780 and A2780 DDP cell lines and primary ovarian cancer cell cultures. β-actin is used as a loading control. c MiR-634 overexpression lowers mRNA levels of CCND1, GRB2, ERK2, RSK1 and RSK2 in the A2780 DDP cell lines 48 h after transfection. Expression was normalized to HPRT and GAPDH expression. Depicted are average values ± SD ( n = 2). The mRNA level in cells transfected with scrambled mimic is set at 100 %. d MiR-634 binds directly to elements of the 3’UTR of CCND1, GRB2, ERK2 and RSK2. A2780 DDP cells were transfected with a scrambled mimic or a miR-634 mimic. After 8 h, the same cells were transfected with a Renilla luciferase reporter construct (psiCHECK™-2) containing a region of 500 bp surrounding the predicted target sites (Additional file : Figure S7), or a luciferase construct in which the miR-634 binding sites were mutated. Since RSK2 contains both a canonical and a non-canonical miR-634 binding site, one construct was generated that contained a mutation in the canonical binding site (RSK2_2 mut 1) and another construct that contained mutations in both the canonical and non-canonical site (RSK2_2 mut 2). The Renilla luciferase activity was measured and normalized using the Firefly luciferase activity. For each construct, the relative luciferase activity of cells transfected with the scrambled mimic was set at 1. Depicted are the average values ± SD ( n = 3). * p < 0.05, ** p < 0.01

Article Snippet: Specific proteins were detected with antibodies against mouse GRB2 (BD Pharmingen, 610111), mouse RSK2 (Santa Cruz, Heerhugowaard, The Netherlands, SC-9986), rabbit ERK1/2 (Cell signaling, Leiden, The Netherlands, #9102), rabbit RSK1 (Santa Cruz, SC-231), rabbit CCND1 (Thermoscientific, Etten-Leur, The Netherlands, RM-9104-S) and mouse anti-β-actin (Sigma, A5441).

Techniques: Derivative Assay, Transfection, Control, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Luciferase, Construct, Binding Assay, Generated, Mutagenesis, Activity Assay