Journal: Molecular and Cellular Biology
Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation
Figure Lengend Snippet: KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.
Article Snippet: Western blot analysis was developed using the following primary and secondary antibodies (antibodies were from Cell Signaling unless otherwise noted): anti-p53 (Ab-7; Calbiochem) (1:2,500), anti-p19ARF (Abcam) (1:400), anti-p15INK4b (Biosource) (1:400), anti-MEK1/2 (1:1,000), anti-phospho-MEK1/2 (1:1,000), anti-phospho-ERK1/2 (1:1,000), anti-Ras (EMD-Biosciences) (1:1,000), anti-caveolin-1 (610059; BD Transduction Laboratories) (1:1000), anti-KSR1 (H-70; Santa Cruz) (1:500), anti-phospho-Ser338 c-Raf (1:500), anti-c-Raf (610151; BD Transduction Laboratories) (1:500), anti-B-Raf (H-145; Santa Cruz) (1:1,000), anti-peroxisome proliferator-activated receptor γ coactivator 1α (anti-PGC-1α) (H-300; Santa Cruz) (1:1,000), anti-estrogen-related receptor α (anti-ERRα) (V-19; Santa Cruz) (1:1,000), and anti-β-actin (Sigma) (1:3,000).
Techniques: Expressing, Binding Assay, Plasmid Preparation, Immunoprecipitation, Proximity Ligation Assay, Negative Control, Generated, Fluorescence