Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: Association of SRSF2 with microtubule proteins. A , I-graph of module 16 (M16) representing hub proteins and corresponding gene symbols as nodes. Node size and edges ( gray ) are reflective of the degree of intramodular connectivity in WGCNA. SRSF2 ( green ) and cytoskeletal-associated proteins ( red ) are highlighted. B , representative western blot of an immunoprecipitation (IP) of recombinant SRSF2-myc in cytoplasm and nucleoplasm extracts isolated from HEK293 cells ( n = 3 replicates per fraction). Co-IP complexes were blotted for α-tubulin (TUBA1A) and β-tubulin (TUBB8). C and D , ICC staining of HEK293 cells for hypophosphorylated SRSF2 (hypoSRSF2) ( green ) and both TUBA1A ( red ; C ) and TUBB8 ( red ; D ) in vehicle-cells or 50 μM SRPIN340-treated cells.

Article Snippet: Approximately 20 μl of Protein A Sepharose 4B beads (catalog no. 101042, Invitrogen) was washed twice in immunoprecipitation (IP) buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 0.5% (v/v) NP-40, 0.5% (v/v) CHAPS, HALT phosphatase inhibitor cocktail (1:100, Thermo Fisher 87786)], then blocked in 0.1 mg/ml bovine serum albumin (Thermo Fisher 23209), then washed an additional three times in IP buffer.

Techniques: Western Blot, Immunoprecipitation, Recombinant, Isolation, Co-Immunoprecipitation Assay, Staining