Fig 2. RGS12 expression is observed in cytosolic puncta within the myoblastic C2C12 cell line, but localized in the nucleus in the poorly differentiating human RD cell line.
(A) Immunoblotting (IB) of cell lysates from all three cell types indicates RGS12 protein expression, as detected using a rabbit anti-RGS12 polyclonal antibody previously described [7]; β-tubulin protein levels were also examined by immunoblotting in parallel as a loading control. (B) Cultures of the poorly differentiating, human rhabdomysarcoma RD cell line and the more-easily differentiated, mouse C2C12 cell line were separately fixed and stained with DAPI; RGS12 protein was detected by indirect immunofluorescence using UNC60-26.2.1. Panels (i-vi) represent: (i, iv) anti-RGS12 antibody detection with Alexa-fluor-594 secondary antibody; (ii, v) DAPI nuclear stain; and, (iii, vi) overlay of both images. (C) Endogenous RGS12 localizes to early (APPL1-positive) endosomes within C2C12 cells. C2C12 cell line cultures (4 x 105 cells/well) were maintained in growth medium (DMEM containing 10% fetal bovine serum [FBS]) for two days. Cells were then fixed in paraformaldehyde, permeabilized, and stained with primary mouse monoclonal antibody UNC60-26.2.1 and secondary Alexa-fluor-594 anti-mouse antibody, alone or in combination with primary anti-APPL1 or -Rab9 rabbit polyclonal antibodies followed by Alexa-fluor-488 secondary anti-rabbit antibody. Overlays in panels (vii-ix) are absent the DAPI nuclear stain (blue) to highlight lack of overlap between RGS12 and Rab9 signals. (D) RGS12 N-terminus binds to select phosphatidylinositides in a lipid dot-blot protein overlay assay. A “PIP Strip” nitrocellulose membrane pre-spotted with the indicated phospholipid species was probed with 20 μg/mL of GST alone, recombinant GST-RGS12 protein (amino-acids 9–406 spanning PDZ and PTB domains; “WT”), or GST-RGS12(aa 9–406) protein with alanine substitutions to four arginines in the PTB domain (Arg-255, -260, -262, and -308; “4R→A”) previously predicted by electrostatic contouring [61] to be involved in phospholipid binding. After extensive washing, the binding of protein to phospholipid spots was detected by chemiluminescence using anti-GST mouse monoclonal antibody and anti-mouse-horseradish peroxidase conjugated secondary antibody. Lipid abbreviations: LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.