mouse anti tubulinβ  (Developmental Studies Hybridoma Bank)

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: Myc tag (16286-1-AP) rabbit, RSK2 (23762-1-AP) rabbit, β tubulin (66240-1-Ig) mouse antibodies were purchased from Proteintech.

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