Journal: Molecular Biology of the Cell

Article Title: h3/Acidic Calponin: An Actin-binding Protein That Controls Extracellular Signal-regulated Kinase 1/2 Activity in Nonmuscle Cells

doi: 10.1091/mbc.E09-06-0451

Figure Lengend Snippet: Interaction studies with h3/acidic CaP, ERK1/2, and PKCα. (A) REF52.2 whole cell lysates were subjected to coimmunoprecipitation by using the rabbit polyclonal anti-ERK1/2 antibody coupled to protein A agarose (lane 3). For negative control, lysates were incubated with rabbit IgG coupled to protein A agarose (lane 2). The immunoprecipitated proteins were separated on an 8.5% SDS-PAGE. A lysate input control (10 μg of total protein) was loaded in lane 1. Arrows indicate the positions of PKCα, ERK1, ERK2, and aCaP. A typical Western blot is shown here, and the experiment was repeated three times. (B) Lysates of REF52.2 cells were incubated with the specific h3/acidic CaP antibody (lane 3) cross-linked to protein G-coupled Dynabeads. Lysates incubated with the GFP-antibody cross-linked to protein G-coupled Dynabeads served as negative control (lane 2). Proteins were eluted from the beads and subsequently analyzed by Western blotting. Ten micrograms of total protein of the lysate was loaded as input (lane 1). Arrows indicate the positions of PKCα, ERK1, ERK2, and aCaP. A typical Western blot is shown here, and the experiment was repeated three times. (C) Recombinant h3/acidic CaP-CBD (aCaP-CBD; lane 2), h1CaP-CBD (lane 3), or the CBD tag alone (lane 1), bound to chitin beads were used in an in vitro pull-down assay to probe for direct interaction with recombinant ERK2 protein. Precipitated proteins were eluted from the chitin beads and analyzed by Western blotting. The membrane was immunostained with an anti-CBD antibody and the polyclonal anti-ERK1/2 antibody.

Article Snippet: The membranes were incubated either with the rabbit polyclonal anti-h3/acidic CaP antibody (calponin 3 H-55; Santa Cruz Biotechnology) at 1:2000 dilution, the mouse monoclonal anti-h1CaP antibody (calponin clone hCP; Sigma-Aldrich) at 1:100,000 dilution, the mouse monoclonal anti-h2CaP antibody (h2-calponin CP21; Hossain et al. , 2005 ) at 1:1000 dilution, the rabbit polyclonal anti-ERK1/2 antibody (ERK1/2 CT; Millipore) at 1:500 dilution, the mouse monoclonal anti-phospho-ERK1/2 antibody (p44/42 MAPK [Thr202/Tyr204] E10; Cell Signaling Technology) at 1:2000 dilution, the mouse monoclonal anti-caldesmon antibody (Abcam) at 1:500 dilution, the rabbit polyclonal anti-phospho-caldesmon (S789) antibody (Millipore) at 1:500 dilution, the mouse monoclonal anti-α-tubulin antibody (Sigma-Aldrich) at 1:5000 dilution, the rabbit polyclonal anti-PAN actin antibody (Cytoskeleton, Denver, CO) at 1:2000 dilution or the mouse monoclonal anti-CBD antibody (New England Biolabs) at 1:2000 dilution.

Techniques: Negative Control, Incubation, Immunoprecipitation, SDS Page, Western Blot, Recombinant, In Vitro, Pull Down Assay